Primase promotes the competition between transcription and replication on the same template strand resulting in DNA damage

Zhang, Weifeng; Yang, Zhuo; Wang, Wenjie; Sun, Qianwen

doi:10.1038/s41467-023-44443-0

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Published: 02 January 2024

Primase promotes the competition between transcription and replication on the same template strand resulting in DNA damage

Nature Communications volume 15, Article number: 73 (2024) Cite this article

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Abstract

Transcription-replication conflicts (TRCs), especially Head-On TRCs (HO-TRCs) can introduce R-loops and DNA damage, however, the underlying mechanisms are still largely unclear. We previously identified a chloroplast-localized RNase H1 protein AtRNH1C that can remove R-loops and relax HO-TRCs for genome integrity. Through the mutagenesis screen, we identify a mutation in chloroplast-localized primase ATH that weakens the binding affinity of DNA template and reduces the activities of RNA primer synthesis and delivery. This slows down DNA replication, and reduces competition of transcription-replication, thus rescuing the developmental defects of atrnh1c. Strand-specific DNA damage sequencing reveals that HO-TRCs cause DNA damage at the end of the transcription unit in the lagging strand and overexpression of ATH can boost HO-TRCs and exacerbates DNA damage. Furthermore, mutation of plastid DNA polymerase Pol1A can similarly rescue the defects in atrnh1c mutants. Taken together these results illustrate a potentially conserved mechanism among organisms, of which the primase activity can promote the occurrence of transcription-replication conflicts leading to HO-TRCs and genome instability.

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Introduction

Organelles in eukaryotic cells are considered the results of early bacterial endosymbiotic events¹. Chloroplasts, the apparatus for photosynthesis, retain a genome of about 100–200 kb in size that comprises two inverted repeats (IR) separated by a small single-copy (SSC) region and a large single-copy (LSC) region². Chloroplasts utilize nucleus-encoded proteins to conduct replication synchronously. Replication normally initiates from the origin, where DNA helicase separates DNA double helix and creates the replication fork utilizing energy from ATP hydrolysis³. DNA primase recognizes single-stranded DNA (ssDNA) to synthesize RNA oligonucleotides used as primers to initiate DNA replication. DNA polymerase utilizes the RNA primers and adds nucleotides matched to the template strand in the 5′ to 3′ direction⁴. While the leading strand is replicated continuously to the 3′ end of the complementary strand, the lagging strand in the opposite direction can only synthesize Okazaki fragments discontinuously as the replication fork moves forward, which processes require the synthesis of RNA primers by primase⁵.

The replisome of the chloroplast genome shares an evolutionary origin with that in bacteriophage T7^6,7,8, which consists of 4 proteins: the coupled primase–helicase (gp4), the single-stranded DNA binding protein (gp2.5), the DNA polymerase (gp5) and its processivity factor E. coli thioredoxin (trx)^9,10. T7 gp4 protein is central to the replication machinery, it is composed of a zinc-binding domain (ZBD) that recognizes ssDNA template, an RNA polymerization domain (RPD) that adds ribonucleotide, and a helicase domain that unwinds double-stranded DNA¹¹. Most eukaryotes own a homolog of the T7 gp4 protein named Twinkle (T7 gp4-like protein with intramitochondrial nucleoid localization)¹². In metazoan organisms, Twinkle lacks the cysteines critical to zinc coordination in the ZBD domain and residues required for RNA synthesis in the RPD domain¹². Instead, RNA polymerase synthesis transcripts are used as primers for DNA replication in metazoan mitochondria¹³.

The Arabidopsis nucleus-encoded Twinkle homolog (Arabidopsis Twinkle homolog, ATH) is a 709-residue protein that localizes in chloroplasts and mitochondria⁶. Previous studies demonstrated that ATH has both primase and helicase activities in vitro⁶. ATH synthesizes RNA primers from a 5′-(G/C)GGA-3′ template sequence, and RNA oligonucleotides synthesized by ATH can be efficiently used as primers for plant organellar DNA polymerases Pol1A and Pol1B¹⁴. ZBD is typically associated with DNA binding domain¹⁵. In bacteriophage primase, two CXXC elements are essential to metal coordination in the ZBD domain¹⁶. In plant primase, the first CXXC repeat is conserved, but the second CXXC repeat is substituted by a CXRXKC element¹⁴. Previous studies indicate that the H33 and K70 residues located before the second CXXC repeat in T7, and Clostridium difficile primase drive ssDNA template recognition, respectively^16,17, and computer modeling of the ZBD domain in ATH indicated that the side chains of R166, K168, and W162 are in similar orientations than H33 of T7 primase and K70 of C. difficile primase¹⁴ suggesting that the R166, K168, and W162 residues may be pivotal in ssDNA template recognition.

The machinery of DNA replication and RNA transcription operate on the same genome template, and collisions occur when they approach. The orientation of gene transcription relative to the direction of the replication fork determines the pattern of transcription-replication conflicts (TRCs). Studies in Bacillus subtilis and human cells revealed that head-on transcription-replication conflicts (HO-TRCs) induce the formation of stable R-loops and block fork progression, representing a major source of genomic instability^18,19,20. Similar phenomena also happen in plants. Our previous studies revealed that the chloroplast-localized RNase H1 protein AtRNH1C can form a complex with chloroplast-localized DNA Gyrases (AtGyrases) and resolve HO-TRCs and R-loops in the rDNA HO-TRCs regions, thus maintaining genome integrity. Mutation of AtRNH1C leads to the formation of aberrant R-loops at these regions, causing genome breaks in chloroplast and growth defects^20,21. By a reverse genetic screening, we also identified a DNA:RNA helicase, RHON1, as an R-loop resolvase operating in parallel with AtRNH1C to restrict HO-TRC-triggered R-loops and maintain genome integrity in chloroplasts, and the HO-TRC-trigged R-loops can be restricted by controlling the transcriptional activity of plastid-encoded RNA polymerases^20,21.

To uncover mechanisms how organisms could coordinate the transcription and replication with R-loop formation and genome maintenance, we adopted a forward genetic screening strategy to identify suppressors of atrnh1c. Here, we report that the primase of chloroplast genome replication, ATH, is responsible for enhancing HO-TRCs thus leading to R-loop accumulation and genome instability in atrnh1c. A point mutation in the zinc-binding domain (ZBD) weakens the binding of template DNA, and decreases the abilities of primer synthesis and delivery, thus slowing down replication and relieving transcription-replication competition. Over-expression of ATH leads to aberrant R-loop accumulation, which can be attenuated by the over-expression of AtRNH1C synchronously. Strand-specific DNA damage sequencing revealed that transcription-replication competition could introduce single-strand DNA breaks near the end of the transcription units. Furthermore, mutation of DNA polymerase Pol1A also can rescue the defects in atrnh1c by a similar mechanism. As HO-TRCs are commonly present in the genomes of all species, our results demonstrated a likely general mechanism of relaxing strand-specific transcription-replication competition to maintain genome integrity.

Results

A point mutation in ATH rescues the developmental defects of atrnh1c

The chloroplast-localized ribonuclease AtRNH1C plays a key role in maintaining plastid genome stability in Arabidopsis, and atrnh1c displays pale yellow leaves, which is caused by aberrant R-loop accumulation and genome degradation²⁰. To investigate new mechanisms regulating HO-TRCs, we conducted an ethyl methanesulfonate (EMS) mutagenesis screen and identified a suppressor of atrnh1c, named acs1 (atrnh1c suppressor 1). Compared with atrnh1c, acs1 showed the recovery of growth defects and leaf color in all stages of plant development, and the quantum efficiency of photosystem II (Fv/Fm), chlorophyll contents, the number and morphological features of chloroplasts in cells, and plant fresh weight all recovered to normal states (Fig. 1a, b and Supplementary Fig. 1).

With SHORE-map analysis²² using the phenotypic recovered plants, we mapped a G to A point mutation in the fourth exon of At1g30680 that was highly correlated with phenotypic variation (Supplementary Fig. 2a, see methods). At1g30680 encodes a DNA primase-helicase with dual localization potential (chloroplast and/or mitochondria, Fig. 1c), for synthesizing RNA primers during organellar DNA replication⁶. Immunoblot analysis showed that the expression levels of chloroplast proteins RPOB, PetA, and RbcL were significantly reduced in atrnh1c compared to Col-0, while there was no difference in the expression of mitochondrial protein IDH (Supplementary Fig. 2b). In line with the phenotype, the levels of chloroplast proteins in the acs1 double mutant were remarkably recovered to the level of Col-0 (Supplementary Fig. 2b). By constructing complementary vectors and genetic transformation, we confirmed that the mutation in At1g30680 is responsible for the phenotypic recovery in acs1 (Fig. 1a, b and Supplementary Fig. 1).

The ATH protein contains a ZBD domain, an RNA polymerization domain (RPD), and a helicase domain, and the point mutation in At1g30680 leads to an amino acid change (R166K) in the ZBD domain (Fig. 1c). By protein structure prediction of Alphafold, we found that R166 is located at the end of a highly confidently predicted β-sheet (Supplementary Fig. 2c). Previous studies indicated that R166 might be vital for the ssDNA template recognition of ATH¹⁴, and multiple sequence alignment revealed that R166 is conserved across land plants (Fig. 1d). EMSA and ChIP-qPCR assays showed that the R166K mutation in ATH indeed caused a significant reduction in the binding capacity to template DNA (Fig. 1e, f). Further genetic approaches confirmed that the complementary sequence with the point mutation could not complement the phenotype of acs1, confirming that the R166K mutation in ATH determines the phenotypic recovery in acs1 (Supplementary Fig. 2d, e).

To directly assess the effect of the R166K mutation on primase activity, we compared the RNA primer synthesis ability of wild-type ATH and ATH(R166K) in vitro, and the results showed that the R166K mutation decreases primer synthesis (Supplementary Fig. 2g). We also detected the ATH and ATH(R166K) mediated RNA-primed DNA synthesis by Pol1A and Pol1B, the DNA polymerases located in plant organelles. When the wild-type ATH was used as primase in the reactions, the amount of synthesized DNA gradually increased as the amount of ATH increased. However, the DNA synthesis was even hard to detect using ATH(R166K) as the primase with equimolar amounts (Supplementary Fig. 2h). Moreover, we compared the helicase activities of the wild-type protein ATH and the mutant protein ATH(R166K), and found that the R166K mutation could affect the helicase activity of ATH (Supplementary Fig. 2i). In conclusion, these results indicate that the R166K mutation in ATH reduces the primer synthesis and delivery abilities as a primase.

ATH is essential for plant development

To further investigate the functions of ATH, we analyzed its subcellular localization. Using transgenic plants of ATH-GFP, we found that the ATH protein displays a punctate distribution in chloroplasts (Fig. 2a, b). Protoplast transformation or tobacco leaf infection assays also showed that ATH is distributed in a punctiform manner in chloroplasts (Supplementary Fig. 3a, b), and the R166K mutation does not change the distribution pattern of ATH (Fig. S3A). The ATH protein could also be detected in mitochondria (Figs. S3A and 3C), which is consistent with previous results²³. Western blot assays revealed that the ATH protein is mainly localized in the thylakoids of chloroplasts and mitochondria (Fig. 2c). The RNase H1 protein AtRNH1C was also found to be enriched within the thylakoids, where the nucleoids are located²⁰. By protoplast co-transformation, we observed a high co-localization distribution of ATH and AtRNH1C in chloroplasts (Fig. 2d, e). The co-localization of ATH with AtRNH1C on the chloroplast thylakoids implies that there might be some functional correlations between them.

**Fig. 2: ATH is co-localized with AtRNH1C in the thylakoids of chloroplasts.**

Considering the bidirectional localization property of ATH, to exclude the effect of mitochondrial localization of ATH on the phenotype, we replaced the ATH signal peptide with the reported signal peptides of AtRNH1C and RecA1 proteins, which were specifically localized in chloroplasts, and the genetic complementation experiments showed that ATH specifically localized to chloroplasts could also effectively recover the phenotype of acs1 (Supplementary Fig. 3d).

We further observed the tissue distribution of ATH using transgenic complementary plants with GUS (β-glucuronidase) fusion. The ATH protein was mainly distributed in young meristematic tissue regions such as SAM (shoot apical meristem), young leaves, root tips, inflorescences, and young siliques (Supplementary Fig. 4). These tissues are the places where active DNA replication is present, and thus we speculate that this expression property of ATH is highly correlated with its function in DNA replication.

The mutant with the R166K mutation in the Col-0 background (named ath-1) exhibited a growth retardation phenotype in the early growth stage compared to Col-0 (Supplementary Fig. 5a), but with growth, the difference between the two is no longer obvious (Supplementary Fig. 5b). A previous study showed that a T-DNA insertion mutant (SALK_152246) of ATH gene displays a phenotype similar to the wild-type plants (Supplementary Fig. 5b)⁸. We examined the position of T-DNA insertion in the SALK_152246 line by sequencing, and found that the T-DNA insertion occurs in the 5′ UTR region of the ATH gene (Supplementary Fig. 5c, d). RT-qPCR assays also showed that the T-DNA insertion does not affect the ATH expression (Supplementary Fig. 5e). We then tried to create knockout mutants of ATH by the CRISPR-Csa9 system²⁴ to further verify its function. However, we were unable to obtain any ath homozygous knockout mutants, either in Col-0 or atrnh1c background. Instead, we obtained several lines of ath+/− heterozygotes (Supplementary Fig. 6). Therefore, the knockout mutation of ATH might be embryonic lethal. We observed the seeds inside the siliques of ath+/− heterozygous deletion plants and found that some of the seeds were white, turning brown and crumbling as they developed (Fig. 3a). The percentage of these problematic seeds in the siliques was calculated to be approximately 25%, while the seeds in either Col-0 or atrnh1c were completely normal (Fig. 3b). By genotyping, we found that the lethal seeds were ath homozygous (Fig. 3c). Further observation found that the development of problematical seeds stayed at the globular stage (Fig. 3d). These results indicated that ATH is indispensable for plant development and that knockout of ATH leads to embryonic lethality.

**Fig. 3: Knockout of *ATH* leads to embryo lethality.**

We analyzed the phenotypes of the CRISPR/Cas9-generated ath^+/− heterozygous and 1cath^+/− plants, and the 1cath-3^+/− plants displayed partially rescued yellowish phenotypes compared to the atrnh1c mutant, while the phenotype of 1cath-5^+/− plants recovers slightly (Supplementary Fig. 6f). The recovery could be because of the loss of ATH protein levels, as the heterozygous mutants carry only one copy of functional ATH gene (Supplementary Fig. 6g). Furthermore, by S9.6 slot-blot assay, we found a significant decrease of R-loops in the chloroplast of 1cath^+/- plants compared to that of atrnh1c (Supplementary Fig. 6h).

acs1 relieves HO-TRCs and restricts R-loop accumulation

To investigate the mechanism of phenotypic recovery caused by the ATH(R166K) mutation, we first examined the R-loop levels in acs1 by chloroplast S9.6 slot-blot, DRIP-qPCR, and immunostaining. It has been previously reported that S9.6 may not be optimal for immuno-staining to detect RNA:DNA hybrid due to possible interference from dsRNA. However, binding affinity measurements in new studies showed S9.6 exhibits specificity for DNA-RNA hybrid over dsRNA^25,26, and our previous immuno-staining assays in chloroplast also showed dsRNA does not affect the R-loop recognitions specificity of S9.6 antibody^21,27. The results all showed a significant decrease in R-loop levels in acs1 chloroplasts compared to atrnh1c, while R-loop levels in complementary materials were comparable to those in atrnh1c (Fig. 4a–c). As R-loop accumulation triggers chloroplast genome instability in atrnh1c²⁰, we investigated whether the decreased level of R-loops in acs1 could relieve genome degradation in the chloroplast. The PFGE (pulsed-field gel electrophoresis) results showed that consistent with previous results²⁰, the monomeric and oligomeric cpDNA (chloroplast DNA) molecules degraded dramatically in atrnh1c compared with Col-0, and these forms were significantly restored in acs1, with a remarkable decrease in degraded DNA molecules (Fig. 4d). TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) and neutral comet assays also confirmed the decrease in DNA damage in acs1 compared to atrnh1c (Supplementary Fig. 7a, b).

We then analyzed the transcription and replication states in acs1. Bioanalyzer (Agilent 4200) results showed that the mature cp-rRNAs were recovered in acs1 compared to atrnh1c (Fig. 4e). The expression of chloroplast rRNA transcription intermediates and mature rRNAs was also calculated by RT-qPCR, and the results showed that the level of rRNA transcripts in acs1 chloroplasts was significantly elevated compared to that in atrnh1c (Supplementary Fig. 7c). We next applied EdU (5-ethynyl-2′-deoxyuridine) staining to assess the replication state in the chloroplast²¹. As with the phenotypic changes, significant DNA replication stress alleviation was observed in acs1 compared to atrnh1c (Fig. 4f). We then conducted a DAPI staining method to analyze DNA replication states by investigating patterns of nucleoids²⁸. In line with previous results, a large percentage of chloroplasts from atrnh1c owned large extended nucleoids (type III), whereas most nucleoids in acs1 displayed scattered distribution (type I or II, Supplementary Fig. 7d), indicating that the chloroplast genome replication stress was mitigated in acs1 comparing to atrnh1c. Furthermore, 2D gel electrophoresis showed that the replication intermediates of the cp-rDNA region were recovered in acs1 compared with atrnh1c (Supplementary Fig. 7e). Taken together, these results imply that the R166K point mutation of ATH alleviates the excessive accumulation of R-loops in atrnh1c, thereby partially restoring the defects of transcription and replication, and maintaining genomic stability in the chloroplast.

HO-TRCs cause single-strand DNA breakage at the end of transcription units

As HO-TRCs could result in R-loop accumulation and DNA damage, we then analyzed the DNA damage sites in the chloroplast genome by DEtail-seq (DNA End tailing and sequencing), a method we recently developed that can detect 3′ end damage sites with strand-specific information²⁹. Along with this experiment, we also analyzed the distribution of the R-loop in the chloroplast genome by ssDRIP-seq³⁰, together with the binding profile of ATH by ChIP-seq. Compared with Col-0, the strand breaks in the atrnh1c chloroplast genome were significantly increased, while the damages in the acs1 genome were decreased compared to those in atrnh1c, with only high signals present in the high transcription-replication collision regions, where R-loop levels were highly accumulated (Fig. 5a–c). Although the DNA breaks were recovered in the double mutant acs1 compared to atrnh1c, they were still higher than those in Col-0 (Fig. 5a–c, Supplementary Fig. 8a–c). Of particular note, single-strand DNA breaks (SSBs) were dramatically enriched on the strand that templates transcription, especially at the end of transcription units, where high HO-TRCs happened (Fig. 5a–c, Supplementary Fig. 8a–c). This pattern of strand-specific SSBs could also be detected in wild-type Col-0 (Fig. 5a–c, Supplementary Fig. 8a–c), although the level was much lower than that in acs1. These results suggested that the primase ATH is mainly for lagging strand replication, and enhances HO-TRCs thus leading to R-loop accumulation and genome instability through HO-TRCs in high transcription regions of the chloroplast genome (Fig. 5d), and the R166K mutation in ATH slows down DNA replication, relieves HO-TRCs and rescues genome integrity in atrnh1c.

**Fig. 5: HO-TRCs cause single-strand DNA breaks at the end of the transcription units.**

Another noteworthy phenomenon is that the overall distribution pattern of chloroplast DNA breaks in atrnh1c is similar to the pattern of the ATH binding profile (Fig. 5a). Previous studies indicated that RNase H is involved in the removal of RNA primers^31,32,33, and RNA primers were excessively accumulated in RNase H mutants³¹. We hypothesize that in atrnh1c, the inability to remove RNA primers from DNA templates leads to the over-accumulation of short RNA:DNA hybrids, which triggers extensive breaks in the chloroplast genome. To test this speculation, we examined the degradation ability of AtRNH1C for RNA primers in vitro, and the results showed that AtRNH1C was able to efficiently remove small fragments of RNA from the DNA template (Supplementary Fig. 8d). Thus, these results indicated that in chloroplasts, AtRNH1C may also function to scavenge RNA primers generated during DNA replication in addition to degrading transcriptionally generated R-loops (Fig. 5d), and the massive accumulation of RNA primers may be an important trigger for extensive breaks in the chloroplast genome of atrnh1c mutant.

ATH antagonizes R-loop clearance machinery to strengthen HO-TRCs and boost DNA damage

To further prove the working model, we artificially overexpressed ATH in Col-0 and acs1, and both resulted in the pale-yellow young leaves of the plants (Fig. 6a), with the number of chloroplasts in cells and the quantum efficiency of photosystem II (Fv/Fm) significantly reduced (Fig. 6b, d, e). The effects became more pronounced as the plants grew and developed (Supplementary Fig. 9a). Consistent with the phenotype, in the ATH overexpression plants, the R-loops were over-accumulated in chloroplasts (Fig. 6c, f–h), and the stability of the chloroplast genome was significantly reduced (Fig. 6i, Supplementary Fig. 9b), companied by a much higher level of DNA damage (Fig. 6j, Supplementary Fig. 9c). In addition, overexpression of ATH also led to transcription and replication inhibition, showing a decrease in cp-rRNA transcription and DNA replication (Supplementary Fig. 9d–g).

**Fig. 6: Overexpression of ATH boosts HO-TRCs and enhances DNA damage.**

S9.6 immunostaining of ATH-GFP overexpression plants showed that the GFP signals were highly co-localized with the S9.6 signals in the chloroplast nucleotides (Fig. 6k). Furthermore, we analyzed the co-localization of ATH with RNA:DNA hybrids by co-transforming ATH-GFP with inactive AtRNH1C(D222N)-mCherry, and results showed that the co-localization of the two proteins can be seen in the nucleoids (Supplementary Fig. 9h). Overexpression of AtRNH1C in ATH overexpressing plants alleviated the phenotype of yellowish leaves (Supplementary Fig. 10). These results showed that the growth defects caused by overexpression of ATH could be relieved by overexpressing AtRNH1C, further confirming an antagonistic function of primase ATH and R-loop clearance machinery that AtRNH1C involved.

Our previous study found that the R-loop helicase RHON1 is also involved in R-loop clearance to maintain chloroplast genome integrity, and the atrnh1c/rhon1 double mutant (1crhon1) displays more severe phenotypic defects than atrnh1c²¹. To investigate whether the R166K mutation in ATH can also rescue the phenotype of 1crhon1, we crossed the rhon1 and acs1 mutants to generate the rhon1acs1 triple mutant (Supplementary Fig. 11a). Compared with 1crhon1, the plant size, photosystem II efficiency, and the number of chloroplasts per cell of the rhon1acs1 triple mutant were all partially restored (Supplementary Fig. 11a–d). S9.6 slot-blot and immunostaining showed a decrease in the R-loop level in the rhon1acs1 triple mutant compared to 1crhon1 (Supplementary Fig. 11e, f), and the TUNEL assay indicated that the DNA damage in the rhon1acs1 triple mutant was weaker than that of 1crhon1 (Supplementary Fig. 11g). The transcription and replication levels in the rhon1acs1 triple mutant also recovered (Supplementary Fig. 11h, i). All these results indicated that the R166K mutation of ATH can also rescue the phenotype of the 1crhon1 double mutant by restricting HO-TRCs thus relieving R-loop accumulation and HO-TRCs. Naturally, the primase ATH and R-loop clearance machinery are antagonistic to each other to balance HO-TRCs and genome integrity.

Mutation of Pol1A also can rescue the growth defects of atrnh1c by restricting HO-TRCs in chloroplasts

Replication of the chloroplast genome requires the involvement of two DNA polymerases, Pol1A and Pol1B³⁴. Previous studies showed that Pol1B is not only involved in replication but also has functions of DNA repair³⁵, and the double mutant of pol1b and atrnh1c displayed more severe chloroplast genome degradation and growth defects than atrnh1c³⁶. To further investigate the effect of DNA replication with R-loop accumulation and genome stability, we constructed a pol1a and atrnh1c double mutant (1cpol1a) (Supplementary Fig. 12a). Compared with atrnh1c, the growth defects, photosystem II efficiency, chlorophyll contents, and chloroplast number per cell in leaves of the 1cpol1a double mutant were partially restored (Fig. 7a–e). DRIP-qPCR and S9.6 immunostaining also showed the R-loop level in the 1cpol1a double mutant decreased compared to atrnh1c (Fig. 7f, g, and Supplementary Fig. 12b). In line with the phenotype, TUNEL assay, PFGE, and Detail-seq results showed that the DNA damage and genome degradation in 1cpol1a also decreased than that of atrnh1c (Fig. 7h, i, and Supplementary Fig. 12c–e). These results further confirmed that weakening DNA replication could relieve HO-TRCs and maintain genome integrity.

**Fig. 7: Mutation of Pol1A rescues the developmental defects of *atrnh1c* by restricting R-loop accumulation and maintaining genome integrity in chloroplasts.**

Discussion

Transcription and replication are the most essential events of living organisms to sustain life, and they rely on the same genome as the template. According to the coding strand of gene transcription relative to the movement of the replisome, the transcription and replication machinery either move head-on or codirectionally, which determines the pattern of transcription-replication conflicts. HO-TRCs induce R-loops and compromise replication and the expression of head-on genes, thus triggering DNA breaks and genome instability^{37,38,39,40,41}. In bacteria, genomes have evolved to organize the majority of genes expressed codirectionally with replication forks, thus avoiding head-on collisions⁴². However, this is not the case in semiautonomous chloroplasts.

Previous results found that the replication origins in the chloroplast genome are located inside the highly transcribed rDNA regions in the IR regions⁴³. This localization feature creates natural HO-TRCs (Fig. 5d). As the rDNAs are the highest transcribed regions in the chloroplasts, and with two origins located inside, this could induce much higher risks of head-on transcription-replication conflicts. By investigating multiple pathways that restrict HO-TRCs-promoted R-loops, we previously found that AtRNH1C and RHON1 synergistically restrict R-loops and release transcription and replication, thus supervising chloroplast genome integrity and ensuring the normal development of plants^20,21. Here, through a phenotypic suppressor screen of atrnh1c, we found that an R166K mutation in the ZBD domain of the chloroplast-localized primase ATH can rescue the growth defects in atrnh1c. Further investigation revealed that the R166K mutation decreases the RNA primer synthesis and delivery activities, slowing down the replication machinery and relieving HO-TRCs, thus reducing R-loops and DNA damage in the chloroplast genome. These results reveal that the chloroplast primase ATH plays a vital role in R-loop coordination and genome integrity maintenance. Furthermore, mutation of Pol1A, one of the two DNA polymerases in plant organelle, can also lead to similar effects in rescuing the defects in atrnh1c mutant. These results indicate that the HO-TRCs can be mitigated by reducing the DNA replication speed, which may be a common mechanism across species.

By DNA breaks sequencing in the chloroplast, we found the DNA breaks were enriched in the lagging strand, especially at the end of transcription units in Col-0 and acs1. This pattern of strand-specific DNA breaks suggested that the primase ATH, which is mainly for lagging strand replication, enhances R-loop accumulation and genome instability at the HO-TRCs regions. We also found the distribution pattern of DNA breaks in atrnh1c is similar to the binding pattern of ATH protein (Fig.5a). Previous studies revealed that RNA primers were excessively accumulated in RNase H mutants³¹. By testing the ability of AtRNH1C to digesting RNA primers, we found that AtRNH1C was able to efficiently remove small fragments of RNA from DNA template (Supplementary Fig. S8d). Thus, we hypothesize that in chloroplasts, AtRNH1C can also degrade RNA primers from DNA templates during replication. In atrnh1c, the inability to remove RNA primers from DNA templates leads to the over-accumulation of RNA:DNA hybrids, which triggers extensive breaks in the chloroplast genome. Indeed, over-expression of ATH in wild type and acs1 also leads to more DNA breaks in the chloroplast genome and plant growth defects, which can be rescued by over-expression of AtRNH1C synchronously (Fig.6, Supplementary Fig. 9, and Supplementary Fig. 10). These genetic and sequencing results further confirmed the hypothesis that AtRNH1C could remove the RNA primers during replication, especially in the RNA primers enriched lagging strand.

Strand-specific mutations occur in the process of DNA replication and transcription. Discontinuous synthesis of the lagging strand during replication produces a series of Okazaki fragments, the 5′ ends of which have increased levels of nucleotide substitution⁴⁴. Additionally, longer exposure as ssDNA may cause the lagging strand to be more vulnerable to mutagens⁴⁵. Mutational asymmetry also occurs on the transcribed and non-transcribed strands during transcription^46,47,48. In the B cell genome of mammals, localized RNA processing protein complex determines the strand-specific mutations, which catalyze proper antibody diversification^49,50. In the genomes of tumors, transcription-coupled damage on the non-transcribed DNA strand and replication-coupled mutagenesis on the lagging-strand template have been detected, and these widespread asymmetric mutations have been proposed to potentially lead to cancer⁵¹. Our findings of strand-specific single-strand DNA breaks in chloroplast genome provide new insights into the molecular mechanisms of strand-specific mutations that occur in a broad range of diseases.

Previous findings confirm that ATH is a bona fide primase indispensable for plant organellar DNA synthesis resembling gp4 in bacteriophage T7, which is essential for the processive replication of phage DNA¹⁴. Knockout of Twinkle leads to depletion of mitochondrial DNA and lethality in humans and mice^12,52,53. Our work also shows the null mutations of ATH lead to embryo lethality in Arabidopsis. These findings further confirmed the key roles of ATH in DNA replication and genome integrity maintenance of chloroplasts.

Dual localization in chloroplasts and mitochondria is a common feature of many organelle proteins encoded by the nucleus^23,54,55. By subcellular localization analysis and immunoblots, we confirmed that the ATH protein also has bidirectional localization properties. Through further studies, we found that the R166K mutation in ATH can weaken R-loop accumulation and enhance genome stability in the chloroplast genome of the atrnh1c mutant. Since AtRNH1C is a protein specifically localized in chloroplasts, we speculate that the effect of ATH mutation is mainly present in chloroplasts. Immunoblot analysis showed that the expression levels of chloroplast proteins were recovered in acs1 compared to atrnh1c, while there was no obvious difference in the expression of mitochondrial protein IDH (Supplementary Fig. 2b). Previous studies showed that DNA maintenance in mitochondria was mainly through the high levels of homologous recombination (HR)-based replication⁵⁶, and probably initially started the replication with RNA synthesized by RNA polymerase as the primer⁵⁷. In addition, changing the dual localization ATH signal peptide with signal peptides that are specifically localized in chloroplasts could also rescue the phenotype of acs1 (Supplementary Fig. 3d). Thus, the importance and function of ATH in mitochondria remain to be further investigated.

Methods

Plant growth and materials

All Arabidopsis thaliana materials used in this study are in the ecotype Columbia-0 (Col) background. The T-DNA insertion mutants atrnh1c, rhon1, and SALK_152246 were obtained from Nottingham Arabidopsis Stock Centre, UK. Surface-sterilized seeds were sown on 1/2 MS medium and incubated at 4˚C for 2 days for stratification. The plants were grown in the chamber under long-day conditions (day/night cycle of 16/8 h) at 22 °C in white light and 18 °C in the dark as described²⁰. All plant materials are used from 21-day-old seedling leaves that grew on 1/2 MS medium, unless otherwise specifically indicated.

For the complementation experiments, ATH or ATH(R166K), genomic DNA sequence from 1.5-kb upstream of ATG to 500-bp downstream of the stop codon were amplified and cloned into the binary vector pCambia1300, thus generating the acs1^ATH::ATH and acs1^{ATH::ATH(R166K)} vectors. Then the GFP or GUS tags were amplified and fused to the C-termini of ATH to generate the acs1^{ATH::ATH-GFP/GUS} and acs1^{ATH::ATH(R166K) -GFP} vectors. The vectors were constructed using the Fast-Cloning method, and the primers are listed in Supplementary Data 1. Then the construct was transformed into acs1 plants, and transformants were selected using hygromycin antibiotics.

To generate ATH overexpression transgenic plants, the coding sequence of the ATH gene without the stop codon was cloned into the binary vector pEarleyGate202 and fused with GFP or FLAG tags at the C-termini. Then the construct was transformed into Col-0 and acs1 plants, and transformants were selected using hygromycin antibiotics. AtRNH1C overexpression transgenic plants were generated by cloning the coding sequence of AtRNH1C into the binary vector pEarleyGate202 without the stop codon, fused with the HA tag at the C-terminus. Then the construct was transformed into Col-0 and atrnh1c plants, and transformants were selected using kanamycin antibiotics.

To generate the genomic mutation of the ATH and Pol1A gene, the plant CRISPR/Cas9 system was used as previously described^20,24. The sequences of sgRNAs are listed in Supplementary Data 1.

All these vectors were transferred into Agrobacterium tumefaciens strain GV3101 and then transformed into Arabidopsis plants by the floral dip method. Transgenic lines were identified through selection using hygromycin or kanamycin antibiotics and verified by PCR and immunoblotting.

Whole-genome sequencing-based mapping

The atrnh1c suppressor line acs1 was backcrossed to atrnh1c, and the F2 population was grown on MS plates for 14-days. Two hundred seedlings with the green leaf phenotype were collected, and genomic DNA was extracted. The genomic DNA was then submitted to the DNA-sequencing facility for library preparation and sequencing on a NovaSeq 6000 system (Illumina) to generate 100-bp paired-end reads, yielding >20-fold genome coverage. The reads were mapped to a Col-0 reference genome (TAIR10), and the putative single-nucleotide polymorphisms (SNPs) were used as markers to identify regions with atrnh1c across the genome using SHOREmap software²². Only C/G-to-T/A transition (EMS-induced) SNP markers were further considered candidates. The causative mutation within the mapping interval was annotated using the SHOREmap software annotate function.

Chlorophyll fluorescence measurements

Chlorophyll fluorescence was measured using the FluorCam (from the Institute of Botany, Chinese Academy of Sciences). The plants were first dark adapted for 30 min before measurement, and the minimum fluorescence yield (Fo) was measured with measuring light. A saturating pulse of white light was applied to measure the maximum fluorescence yield (Fm). The maximal photochemical efficiency of PSII was calculated based on the ratio of Fv (Fm-Fo) to Fm. For the image analysis, the corresponding data from the plants were normalized to a false-color scale with an assigned extremely high value of 0.8 (red) and a lower value of 0.4 (blue).

Phylogenetic analysis and molecular modeling

The ATH protein sequences were used as queries to search against various species genomes in NCBI with BLASTP. Multiple sequences were submitted to the phylogenetic analysis tool NGPhylogeny.fr with default settings for sequence alignments. For phylogenetic tree construction, the FastME Output Tree was then uploaded to iTOL (version 5) for tree visualization. The 3D predicted structure of ATH was obtained from the AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk).

Expression and purification of recombinant proteins

The full-length CDS of ATH, ATH(R166K), Pol1A, and Pol1B were amplified and cloned into the pGEX-4T vector and expressed in Rosetta (DE3) cells. Rosetta (DE3) cells were grown at 37 °C until the OD600 reached 0.6, and then 0.5 mM IPTG was added. The cells were then held at 18 °C and incubated overnight (16 h) with shaking. After centrifugation at 4000 g, the harvested cells were re-suspended in 1x PBS and sonicated on ice until the suspension became transparent. The supernatant was collected and incubated with GST agarose resins (YEASEN, 20507ES50) at 4 °C for 4 h. The agarose resins were washed four times with 1x PBS and the proteins were eluted with elution buffer (50 mM reduced glutathione in 1x PBS). The quality of GST-ATH and GST-ATHR166K proteins was tested by SDS-PAGE gel, and the protein concentrations were measured with a Bradford Protein Assay Kit (Beyotime, P0006C).

Electrophoretic Mobility Shift Assay (EMSA)

Unlabeled and 3′-FAM-tagged synthetic oligonucleotides were used as probes (sequence: SGGASGGASGGASGGASGGASGGASGGA). The ATH and ATH(R166K) proteins (0.5 µg to 5 µg) were incubated with 20 pmol probes in 1x binding buffer (Beyotime, GS005) for 30 min, then the reaction mixture was separated on 8.5% native PAGE gel and visualized by Typhoon FLA9500.

Template-directed primer synthesis and RNA-primed DNA synthesis

Primase reactions were assayed with 100 nM ssDNA template (5′-(T) ₇GGGA(T) ₇-3′), 100 µM GTP, CTP, UTP, and 10 µCi of [γ-³²P]-ATP (NEG502A) in a buffer containing 40 mM Tris–HCl pH 7.5, 50 mM potassium glutamate, 10 mM MgCl2 and 10 mM DTT. Each primase reaction contained varied amounts of recombinant protein as indicated in the figures. After incubation at 30 °C for 60 min, loading buffer (95% formamide, 0.1% xylene cyanol) was added to the reaction products, and then the products were separated on a 27% denaturing polyacrylamide gel containing 3 M urea. The autoradiographs were exposed to the phosphor screens for 2 days and then scanned using Typhoon FLA9500.

RNA-primed DNA synthesis were assayed with 100 nM ssDNA template (5′-(T) ₅A(T) ₉GGGGA(T) ₁₀-3′), 100 µM NTPs, 100 µM dATP, 100 µM dGTP, 100 µM dTTP, and 10 µCi of [α-³²P]-dCTP (NEG513H) in a same buffer as above. Each primase reaction contained varied amounts of recombinant protein as indicated in the figures. After incubation at 30 °C for 60 min, the reaction products were detected as described above.

Helicase assay

ATH helicase reactions were assayed in a buffer containing 10 mM Tris–HCl pH 8.0, 8 mM MgCl2, 1 mM DTT, and 5 mM ATP. 25 nM or 10 nM 3′-FAM-tagged dsDNA (SGGASGGASGGASGGASGGASGGASGGA) were used as substrate. Each helicase reaction contained varied amounts of recombinant protein as indicated in the figure. After incubation at 37 °C for 2 h, loading buffer (95% formamide, 0.1% xylene cyanol) was added to the reaction products, and then the products were separated on a 9% native polyacrylamide gel and scanned using Typhoon FLA9500.

Protoplast transformation

The CDS of ATH and ATH(R166K) were cloned into pUC19-35S-eGFP. Protoplasts were extracted from 30-day-old leaves and transformed with plasmid-mediated by 20% PEG-Ca. After 16 h of transient expression, protoplasts were observed under fluorescence microscopy (Zeiss, LSM880).

Chloroplast and mitochondrion fractionation

Chloroplast sub-fractionation analysis was performed as previously described⁵⁸ with minor modifications. Briefly, 21-day-old plants were homogenized in CIB buffer (10 mM HEPES–KOH pH 8.0, 150 mM sorbitol, 2.5 mM EDTA pH 8.0, 2.5 mM EGTA pH 8.0, 2.5 mM MgCl₂, 5 mM NaHCO₃, and 0.1% BSA) on ice. The homogenate was further filtered through a double layer of Miracloth and centrifuged at 200 g/4 °C for 3 min. Then the supernatant was transferred to new 50 ml tubes and centrifuged for 10 min at 1200 g/4 °C to obtain intact chloroplasts. The chloroplasts were resuspended in buffer II (0.33 M sorbitol, 5 mM MgCl₂, 2.5 mM EDTA pH 8.0, 20 mM HEPES–KOH pH 8.0) and buffer III (5 mM MgCl₂, 25 mM EDTA pH 8.0, 20 mM HEPES–KOH pH 8.0), successively. After centrifugation at 4 °C, the stroma was in the liquid supernatant, while the sediment contained the thylakoid fraction.

Mitochondrial fractionation was performed as previously described⁵⁵. 21-day-old plants were homogenized in ice-cold grinding buffer (0.3 M sucrose, 25 mM tetrasodium pyrophosphate, 1% (w/v) polyvinylpyrrolidone-40, 2 mM EDTA, 10 mM KH₂PO₄, 1% (w/v) BSA, 20 mM sodium l-ascorbate, 1 mM DTT, 5 mM cysteine, pH 7.5). The homogenate was filtered through 4 layers of Miracloth and centrifuged at 2,500 g/4 °C for 5 min, and the supernatant was then centrifuged at 20,000 g/4 °C for 15 min. The pellet was resuspended in washing buffer (0.3 M sucrose, 10 mM TES, pH 7.5), and repeated 1500 g and 20,000 centrifugation steps. The resulting pellet was gently resuspended in washing buffer and fractionated on a Percoll step gradient (18% to 27% to 50%) by centrifugation at 40,000 g for 45 min. Mitochondria were collected at the 27% to 50% interface and diluted with washing buffer. After centrifugation at 31,000 g/4 °C for 15 min, the mitochondrial pellet was collected for use.

Protein extraction and immunoblot analysis

Total protein was extracted with protein extraction buffer (50 mM Tris-HCl pH 7.4, 154 mM NaCl, 10% glycerol, 5 mM MgCl₂, 1% Triton X-100, 0.3% NP-40, 5 mM DTT, 1 mM PMSF, and protease inhibitor cocktail). Anti-FLAG (Sigma, F1804), anti-GFP (ABclonal, AE012), anti-HA (Beyotime, AF5057), anti-plant-actin (ABclonal, AC009), anti-RPOB (PhytoAB, PHY1701), anti-PetA (PhytoAB, PHY0023), anti-RbcL (Agrisera, AS03037A), anti-PsaA (PhytoAB, PHY0053A), and anti-IDH (PhytoAB, PHY0098A) were used as primary antibodies, and goat anti-mouse (EASYBIO, BE0102) or goat anti-rabbit antibodies (EASYBIO, BE0101) were used as secondary antibodies.

GUS staining

The acs1^ATH::ATH-GUS transformed Arabidopsis plants were incubated with staining buffer (0.1 M K₃[Fe(CN)₆], 0.1 M K₄[Fe(CN)₆], 1 M NaH₂PO₄, 1 M Na₂HPO₄, 0.5 M EDTA (pH 8.0) and 20% methanol) under vacuum for 10 min and then overnight at 37 °C. After staining, the plants were washed three times with 100% alcohol and then photographed.

Seed clearing and observation

Seed clearing was performed as previously described^55,59 with minor modifications. In brief, developing siliques at various stages were fixed in ethanol:acetic acid (9:1) and washed with 70% ethanol. The seeds were isolated, mounted on slides in Hoyer’s medium (glycerol/water/chloral hydrate in a ratio of 1:2:8, v/v/w), and observed under a differential interference contrast (DIC) light microscope (Olympus, BX53).

Chloroplast isolation and chloroplast DNA extraction

Leaves of 21-day-old plants were harvested to extract intact chloroplasts using a chloroplast isolation kit (Invent, CP-011) following the instructions from the manufacturer. Briefly, plant leaves were added to a filter column containing 200 µl cold buffer A, and were gently ground with a grinding rod for 2 min on ice. The filter was capped and centrifuged at 2000 g for 5 min. The pellet was suspended in 200 µl cold buffer B and centrifuged at 2000g for 10 min. The chloroplast was washed twice in CIB buffer before use. The chloroplasts were used for the comet assay, TUNEL assay, and immunofluorescence staining.

For PFGE, Slot-blot, DRIP, and cpChIP, chloroplasts were extracted by grounding plant leaves in 20 ml ice-cold CIB buffer (10 mM HEPES–KOH (pH 8.0), 150 mM sorbitol, 2.5 mM EDTA (pH 8.0), 2.5 mM EGTA (pH 8.0), 2.5 mM MgCl₂, 5 mM NaHCO₃, and 0.1% BSA). The homogenate was filtrated through two layers of Miracloth and centrifuged at 200 g/4 °C for 3 min. Then the supernatant was transferred to new 50 ml tubes and centrifuged for 10 min at 1200 g/4 °C. The pellet was then suspended with cold CIB buffer and was ready for use.

For chloroplast DNA extraction, chloroplasts were lysed in chloroplast DNA extraction buffer (CIB with 1% SDS and proteinase K) at 37 °C overnight with shaking, and then SDS was removed by adding 20 µM KAc. Chloroplast DNA was purified by phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v) and precipitated with an equal volume of isopropyl alcohol at -20 °C overnight. The chloroplast DNA was dissolved in 1x TE.

DAPI staining and immunostaining

For DAPI staining, the intact chloroplast was fixed in 4% paraformaldehyde for 10 min at room temperature. Then the chloroplast was washed three times with 1x PBS and stained with DAPI. The stained chloroplast was observed by a confocal microscope (Zeiss, LSM880).

For immunostaining, the fixed chloroplasts were refixed on PLL-coated slides (CITOGLAS, 188105) with 4% paraformaldehyde for 10 min and then washed three times with 1x PBS. Then the chloroplasts were pretreated with RNase III for 30 min and washed with 1x PBS. The samples were then blocked with blocking buffer (1% BSA, 0.3% Triton X-100 in 1x PBS) for 20 min at room temperature and incubated with 100 µl of S9.6 antibody diluted in blocking solution (1:100) at 4 °C overnight. The slides were washed three times with 1x PBS and incubated with 100ul of secondary antibody for 1 h at room temperature. Then, the cells were washed three times washing with 1x PBS. 20ul of protective agents (Southern Biotech, 0100-20) were added to the slides and covered with cover glass. The observation was performed by a confocal microscope (Zeiss, LSM880).

Slot-blot hybridization analysis

The detailed Slot-Blot assay was described previously²⁰ with minor modifications. Briefly, 5 μg chloroplast DNA extracted from different samples was treated with 1 U of RNase III (NEB, M0245S) at 37 °C for 30 min, then purified with phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v) and precipitated with an equal volume of isopropyl alcohol. The purified DNA was spotted on Hybond N+ membrane using a slot-blot apparatus and vacuum suction. The membrane was then crosslinked and blocked in 5% milk-TBST, and detected by S9.6 antibody (DNA:RNA hybrid-specific antibody).

Chloroplast chromatin immunoprecipitation (cpChIP)

cpChIP was performed as described previously³⁶. The chloroplasts were cross-linked with 1% formaldehyde for 10 min, and the cross-linking reaction was stopped by adding 150 µl 1 M glycine and incubating for 10 min. Then the cross-linked chloroplasts were washed twice with CIB and lysed in lysis buffer (50 mM Tris-HCl (pH 7.6), 0.15 M NaCl, 1 mM EDTA (pH 8.0), 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate). Chloroplast DNA was sheared with sonication into fragments of ∼500-bp. The supernatant was incubated with an anti-GFP antibody (abcam, ab290) overnight at 4 °C. Plants without a GFP tag were adopted as the negative control. ChIP-qPCR was performed using the immunoprecipitated DNA and input DNA. Primers corresponding to four rDNA regions were used for detection and are listed in Supplementary Data 1.

DNA:RNA hybrid immunoprecipitation (DRIP)

5 μg of chloroplast DNA was fragmented with 5 U of DdeI (NEB, R0175V), MseI (NEB, R0525S), RsaI (NEB, R0167V), and AluI (NEB, R0137V) at 37 °C for 12 h. RNase H pretreated atrnh1c cpDNA was used as the negative control. The fragmented DNA was then purified by phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v) and precipitated with an equal volume of isopropyl alcohol. 2 µg of purified fragment DNA with or without RNase H treatment was incubated with 10 µg of S9.6 antibody overnight at 4 °C. Samples were further incubated with 50 µl Protein G beads (Invitrogen, 10004D) for 4 h at 4 °C. The immunoprecipitated DNA was purified as mentioned above. The primers used for DRIP-qPCR are listed in Supplementary Data 1.

DEtail-seq

DEtail-seq assay was described previously²⁹ with minor modifications. Briefly, the chloroplasts were embedded in low-melting-point agarose and lysed in lysis buffer as described above. Agarose plugs were then washed in 1x TE buffer 4 times at 37 °C, with the first two washes containing 1 mM PMSF. Then, agarose plugs were cut into small pieces and lysed in 10 μg/ml RNase A at 37 °C overnight. The lysed agarose pieces were then washed in 1x TE buffer 5 times at 37 °C, followed by two washes with 300 μl 1x CutSmart (NEB, B7204S) at 37 °C. The agarose pieces were then incubated with 3 μl I-CeuI (NEB, R0699S) in 150 μl 1x CutSmart Buffer at 37 °C for 12 h and washed 3 times with 1x TE buffer. Then the T7 ligation process was conducted by adding 65 μl (T7 Buffer 8 μl, T7 Adapter 5 μl, T7 Enzyme Mix II 6 μl, Low-EDTA TE 46 μl) T7 Tailing & Ligation solution (ABclonal, RK20228) and incubating at 37 °C for 12 h. The DNA in agarose was purified using a DNA gel purification kit (Magen, D2111-02), and fragmented to ~250 bp using a focused ultrasonicator (Covaris, S220). The following library preparation steps were conducted according to the manual (ABclonal, RK20228).

In vitro removal of RNA primer by AtRNH1C

The assay was performed as previously described⁵⁵. 100 nM FAM-labeled RNA:DNA hybrid (8-bp) was used as the substrate. The reaction was performed in a buffer containing 50 mM KCl, 4 mM MgCl2, 20 mM HEPES-KOH pH 7.0, 4% glycerol, 50 μg/ml BSA, and 1 mM DTT. After incubation with 100 to 400 nM purified GST-AtRNH1C or GST-GFP proteins for 30 min, the reactions were stopped by 20 mM EDTA. The products were separated on a 12% native polyacrylamide gel and then scanned using Typhoon FLA9500.

Single-cell gel electrophoresis assay (Comet assay)

The comet assay was performed as previously described²⁰. Briefly, 10 μl of intact chloroplasts were mixed with 90 μl of LM Agarose at 37 °C, and 50 μl of each sample was added to the CometSlide. Slides were incubated at 4 °C in the dark for 20 min for solidification and then incubated in lysis solution (R&D, 4250-050-01) overnight at 4 °C in the dark. The slides were further incubated in neutral electrophoresis buffer (50 mM Tris and 150 mM sodium acetate, pH 9.0) for 30 min. For electrophoresis, the slides were run at 1 V/cm for 20 min in neutral electrophoresis buffer. Slides were then incubated in DNA precipitation solution (1 M NH₄Ac in 95% ethanol) for 30 min and 70% ethanol for another 30 min. After drying at 37 °C, the slides were stained with SYBR Green for 30 min followed by a water wash. Samples were visualized by epifluorescence microscopy (Olympus, BX53) at 488 nm. The OpenComet tool⁶⁰ launched from ImageJ software was used for the analysis and quantification of the results.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay)

TUNEL assay was performed using a TUNEL Apoptosis Assay Kit-FITC (7sea, AT005-1) according to the manufacturer’s instructions. Briefly, the intact chloroplasts were extracted and fixed onto PLL-coated slides. Then the samples were blocked by blocking buffer (1% BSA, 0.3% Triton X-100 in 1x PBS) for 20 min at room temperature. The slides were rinsed with 1x PBS three times and then incubated with TdT reaction mixture for 1 h at 37 °C. After three washes with 1x PBS, chlorophyll autofluorescence and TUNEL fluorescence were captured under confocal microscopy (Zeiss, LSM880). The fluorescence intensity was quantified by ImageJ software.

EdU labeling of Arabidopsis chloroplast

21-day-old plants were transferred to liquid 1/2 MS medium with 20 μM EdU (BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488) and grown in the chamber for 17 h. Then the plants were washed with 1x PBS three times and fixed in 4% paraformaldehyde in 1x PBS for 10 min. After being fixed, the plants were washed twice with 1x PBS, and a MinuteTM Chloroplast Isolation Kit (CP-011, Invent) was used to extract intact chloroplasts. The chloroplasts were fixed in 4% paraformaldehyde again on PLL-coated slides for 30 min and covered with 0.3% Triton X-100 in 1x PBS for 20 min at room temperature. Then the slides were incubated with 50 μl Click-iT® reaction cocktail (43 μl of 1x Click-iT® EdU reaction buffer, 2 μl of CuSO₄, 0.1 μl of Alexa Fluor® azide, and 5 μl of 1XClick-iT® EdU buffer additive) for 1 h at room temperature. Then, the cells were washed three times with 1x PBS, and 10 µl protective agent (Southern Biotech, 0100-20) covered with cover glass. The observation was performed by a confocal microscope (Zeiss, LSM880).

Two-dimensional gel electrophoresis of replication intermediates (2D-gel)

Two-dimensional gel electrophoresis was performed as described previously²⁰. A total of 20 μg cpDNA from each sample was digested with 10 U of the restriction enzymes (AseI and BglI) and precipitated with isopropanol. For the first-dimension gel electrophoresis, the digested DNA was loaded onto a 0.3% agarose gel without ethidium bromide, in 0.5x TBE buffer at 0.7 V/cm for 30 h. The second-dimension gel electrophoresis was performed in a 1% agarose gel containing 0.3 μg/ml ethidium bromide, at 6 V/cm for 5 h at 4 °C. DNA was transferred onto Hybond N+ membrane (GE, RPN303B) according to standard DNA gel blotting methods. The blots were hybridized to radiolabeled probes labeled with [α-³²P]-dCTP (NEG513H) using a Random Primer DNA Labeling Kit Ver. 2 (Takara, 6045). The autoradiographs were exposed to the phosphor screens for 10 days and scanned by Typhoon FLA9500.

Pulsed-field gel electrophoresis (PFGE)

The chloroplasts suspended in CIB were mixed with 1% low-melting point agarose (Promega, V2111) dissolved in TE buffer (1:1, v/v) at 37 °C. The plugs were solidified at 4 °C for 30 min and then lysed in lysis buffer (1% sarkosyl, 0.45 M EDTA, 10 mM Tris-HCl (pH 8.0) and 2 mg/mL proteinase K) at 48 °C for 16 h with shaking. The lysis buffer was exchanged three times. Agarose plugs were then washed in 1x TE buffer 6 times at 4 °C, with the first two washes containing 1 mM PMSF, filled into 1% agarose gel, and subjected to electrophoresis in 0.5x TBE for 42 h at 14 °C using a CHEF Mapper XA system (Bio-Rad). A Lambda Ladder (New England Biolabs; N0341) was used to indicate the molecular weight. The detailed electrophoresis parameters were 5 to 120 s of pulse time at 4.5 V/cm. After EtdBr staining and photography, the gel was blotted onto a Hybond N+ membrane (GE, RPN303B) according to standard DNA gel blotting methods. A 505-bp fragment of the chloroplast rbcL gene (55677–56181) was labeled with [α-³²P]-dCTP (NEG513H) using Random Primer DNA Labeling Kit Ver. 2 (Takara, 6045) and used as a probe for hybridization. The autoradiographs were exposed to the phosphor screens for 5 days and then scanned using Typhoon FLA9500.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The sequencing data generated in this study have been deposited into NCBI’s Gene Expression Omnibus (GEO) database and are accessible through the GEO Series accession number GSE215443. Source data are provided with this paper.

Change history

15 January 2024
A Correction to this paper has been published: https://doi.org/10.1038/s41467-024-44904-0

References

Reyes-Prieto, A., Weber, A. P. & Bhattacharya, D. The origin and establishment of the plastid in algae and plants. Annu Rev. Genet. 41, 147–168 (2007).
Article CAS PubMed Google Scholar
Johnston, I. G. Tension and resolution: dynamic, evolving populations of organelle genomes within plant cells. Mol. Plant 12, 764–783 (2019).
Article CAS PubMed Google Scholar
Lionnet, T., Spiering, M. M., Benkovic, S. J., Bensimon, D. & Croquette, V. Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism. Proc. Natl Acad. Sci. USA 104, 19790–19795 (2007).
Article ADS CAS PubMed PubMed Central Google Scholar
Seeman, N. C. DNA replication and recombination. Nature 421, 427–431 (2003).
Article ADS MathSciNet PubMed Google Scholar
Lee, J. B. et al. DNA primase acts as a molecular brake in DNA replication. Nature 439, 621–624 (2006).
Article ADS CAS PubMed Google Scholar
Diray-Arce, J., Liu, B., Cupp, J. D., Hunt, T. & Nielsen, B. L. The Arabidopsis At1g30680 gene encodes a homologue to the phage T7 gp4 protein that has both DNA primase and DNA helicase activities. BMC Plant Biol. 13, 36 (2013).
Article CAS PubMed PubMed Central Google Scholar
Towle-Weicksel, J. B., Cao, Y., Crislip, L. J., Thurlow, D. L. & Crampton, D. J. Chimeric proteins constructed from bacteriophage T7 gp4 and a putative primase-helicase from Arabidopsis thaliana. Mol. Biol. Rep. 41, 7783–7795 (2014).
Article CAS PubMed Google Scholar
Morley, S. A. et al. Arabidopsis thaliana organelles mimic the T7 phage DNA replisome with specific interactions between Twinkle protein and DNA polymerases Pol1A and Pol1B. BMC Plant Biol. 19, 241 (2019).
Article PubMed PubMed Central Google Scholar
Hamdan, S. M. & Richardson, C. C. Motors, switches, and contacts in the replisome. Annu. Rev. Biochem. 78, 205–243 (2009).
Article CAS PubMed Google Scholar
Lee, S. J. & Richardson, C. C. Choreography of bacteriophage T7 DNA replication. Curr. Opin. Chem. Biol. 15, 580–586 (2011).
Article CAS PubMed PubMed Central Google Scholar
Kulczyk, A. W. & Richardson, C. C. Molecular interactions in the priming complex of bacteriophage T7. Proc. Natl Acad. Sci. USA 109, 9408–9413 (2012).
Article ADS CAS PubMed PubMed Central Google Scholar
Spelbrink, J. N. et al. Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria. Nat. Genet. 28, 223–231 (2001).
Article CAS PubMed Google Scholar
Wanrooij, S. et al. Human mitochondrial RNA polymerase primes lagging-strand DNA synthesis in vitro. Proc. Natl Acad. Sci. USA 105, 11122–11127 (2008).
Article ADS CAS PubMed PubMed Central Google Scholar
Peralta-Castro, A., Baruch-Torres, N. & Brieba, L. G. Plant organellar DNA primase-helicase synthesizes RNA primers for organellar DNA polymerases using a unique recognition sequence. Nucleic Acids Res. 45, 10764–10774 (2017).
Article CAS PubMed PubMed Central Google Scholar
Laity, J. H., Lee, B. M. & Wright, P. E. Zinc finger proteins: new insights into structural and functional diversity. Curr. Opin. Struct. Biol. 11, 39–46 (2001).
Article CAS PubMed Google Scholar
Kusakabe, T., Hine, A. V., Hyberts, S. G. & Richardson, C. C. The Cys4 zinc finger of bacteriophage T7 primase in sequence-specific single-stranded DNA recognition. Proc. Natl Acad. Sci. USA 96, 4295–4300 (1999).
Article ADS CAS PubMed PubMed Central Google Scholar
van Eijk, E. et al. Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile. Open Biol. 6, (2016).
Lang, K. S. et al. Replication-transcription conflicts generate R-loops that orchestrate bacterial stress survival and pathogenesis. Cell 170, 787–799.e18 (2017).
Article CAS PubMed PubMed Central Google Scholar
Hamperl, S., Bocek, M. J., Saldivar, J. C., Swigut, T. & Cimprich, K. A. Transcription-replication conflict orientation modulates R-loop levels and activates distinct DNA damage responses. Cell 170, 774–786.e19 (2017).
Article CAS PubMed PubMed Central Google Scholar
Yang, Z. et al. RNase H1 cooperates with DNA gyrases to restrict R-loops and maintain genome integrity in Arabidopsis chloroplasts. Plant Cell 29, 2478–2497 (2017).
Article CAS PubMed PubMed Central Google Scholar
Yang, Z., Li, M. & Sun, Q. RHON1 co-transcriptionally resolves R-loops for arabidopsis chloroplast genome maintenance. Cell Rep. 30, 243–256.e5 (2020).
Article CAS PubMed Google Scholar
Sun, H. & Schneeberger, K. SHOREmap v3.0: fast and accurate identification of causal mutations from forward genetic screens. Methods Mol. Biol. 1284, 381–395 (2015).
Article CAS PubMed Google Scholar
Carrie, C. et al. Approaches to defining dual-targeted proteins in Arabidopsis. Plant J. 57, 1128–1139 (2009).
Article CAS PubMed Google Scholar
Wang, Z. P. et al. Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biol. 16, 144 (2015).
Article PubMed PubMed Central Google Scholar
Bou-Nader, C., Bothra, A., Garboczi, D. N., Leppla, S. H. & Zhang, J. W. Structural basis of R-loop recognition by the S9.6 monoclonal antibody. Nat. Commun. 13, (2022).
Li, Q. et al. Cryo-EM structure of R-loop monoclonal antibody S9.6 in recognizing RNA:DNA hybrids. J. Genet. Genomics 49, 677–680 (2022).
Article PubMed Google Scholar
Li, Q. et al. DNA polymerase ε harmonizes topological states and R-loops formation to maintain genome integrity in Arabidopsis. Nat. Commun. 14, (2023).
Kobayashi, Y. et al. Holliday junction resolvases mediate chloroplast nucleoid segregation. Science 356, 631–634 (2017).
Article ADS CAS PubMed Google Scholar
Xu, W. et al. DEtail-seq is an ultra-efficient and convenient method for meiotic DNA break profiling in multiple organisms. Sci. China Life Sci. 66, 1392–1407 (2023).
Xu, W. et al. The R-loop is a common chromatin feature of the Arabidopsis genome. Nat. Plants 3, 704–714 (2017).
Article CAS PubMed Google Scholar
Kitani, T., Yoda, K., Ogawa, T. & Okazaki, T. Evidence that discontinuous DNA replication in Escherichia coli is primed by approximately 10 to 12 residues of RNA starting with a purine. J. Mol. Biol. 184, 45–52 (1985).
Article CAS PubMed Google Scholar
Uhler, J. P. & Falkenberg, M. Primer removal during mammalian mitochondrial DNA replication. DNA Repair (Amst.) 34, 28–38 (2015).
Article CAS PubMed Google Scholar
Williams, J. S., Lujan, S. A. & Kunkel, T. A. Processing ribonucleotides incorporated during eukaryotic DNA replication. Nat. Rev. Mol. Cell Biol. 17, 350–363 (2016).
Article CAS PubMed PubMed Central Google Scholar
Mori, Y. et al. Plastid DNA polymerases from higher plants, Arabidopsis thaliana. Biochem. Biophys. Res. Commun. 334, 43–50 (2005).
Article CAS PubMed Google Scholar
Parent, J. S., Lepage, E. & Brisson, N. Divergent roles for the two PolI-like organelle DNA polymerases of Arabidopsis. Plant Physiol. 156, 254–262 (2011).
Article CAS PubMed PubMed Central Google Scholar
Wang, W. et al. RNase H1C collaborates with ssDNA binding proteins WHY1/3 and recombinase RecA1 to fulfill the DNA damage repair in Arabidopsis chloroplasts. Nucleic Acids Res. 49, 6771–6787 (2021).
Article CAS PubMed PubMed Central Google Scholar
Crossley, M. P., Bocek, M. & Cimprich, K. A. R-loops as cellular regulators and genomic threats. Mol. Cell 73, 398–411 (2019).
Article CAS PubMed PubMed Central Google Scholar
Garcia-Muse, T. & Aguilera, A. R loops: from physiological to pathological roles. Cell 179, 604–618 (2019).
Article CAS PubMed Google Scholar
Gomez-Gonzalez, B. & Aguilera, A. Transcription-mediated replication hindrance: a major driver of genome instability. Genes Dev. 33, 1008–1026 (2019).
Article CAS PubMed PubMed Central Google Scholar
Bayona-Feliu, A. & Aguilera, A. The role of chromatin at transcription-replication conflicts as a genome safeguard. Biochem. Soc. Trans. 49, 2727–2736 (2021).
Article CAS PubMed Google Scholar
Brickner, J. R., Garzon, J. L. & Cimprich, K. A. Walking a tightrope: the complex balancing act of R-loops in genome stability. Mol. Cell 82, 2267–2297 (2022).
Article CAS PubMed PubMed Central Google Scholar
Merrikh, H. Spatial and temporal control of evolution through replication-transcription conflicts. Trends Microbiol 25, 515–521 (2017).
Article CAS PubMed PubMed Central Google Scholar
Kunnimalaiyaan, M. & Nielsen, B. L. Fine mapping of replication origins (ori A and ori B) in Nicotiana tabacum chloroplast DNA. Nucleic Acids Res. 25, 3681–3686 (1997).
Article CAS PubMed PubMed Central Google Scholar
Reijns, M. A. M. et al. Lagging-strand replication shapes the mutational landscape of the genome. Nature 518, 502–506 (2015).
Article ADS CAS PubMed PubMed Central Google Scholar
Yu, C. et al. Strand-specific analysis shows protein binding at replication forks and pcna unloading from lagging strands when forks stall. Mol. Cell 56, 551–563 (2014).
Article CAS PubMed PubMed Central Google Scholar
Jinks-Robertson, S. & Bhagwat, A. S. Transcription-associated mutagenesis. Annu. Rev. Genet. 48, 341–359 (2014).
Article CAS PubMed PubMed Central Google Scholar
Georgakopoulos-Soares, I. et al. Transcription-coupled repair and mismatch repair contribute towards preserving genome integrity at mononucleotide repeat tracts. Nat. Commun. 11, (2020).
Polak, P., Querfurth, R. & Arndt, P. F. The evolution of transcription-associated biases of mutations across vertebrates. BMC Evol. Biol. 10, (2010).
Lim, J. et al. Nuclear proximity of Mtr4 to RNA exosome restricts DNA mutational asymmetry. Cell 169, 523–537 (2017).
Article CAS PubMed PubMed Central Google Scholar
Laffleur, B. et al. Noncoding RNA processing by DIS3 regulates chromosomal architecture and somatic hypermutation in B cells. Nat. Genet. 53, 230–242 (2021).
Article CAS PubMed PubMed Central Google Scholar
Haradhvala, N. J. et al. Mutational strand asymmetries in cancer genomes reveal mechanisms of DNA damage and repair. Cell 164, 538–549 (2016).
Article CAS PubMed PubMed Central Google Scholar
Wanrooij, S. et al. Twinkle and POLG defects enhance age-dependent accumulation of mutations in the control region of mtDNA. Nucleic Acids Res. 32, 3053–3064 (2004).
Article CAS PubMed PubMed Central Google Scholar
Tyynismaa, H. et al. Mutant mitochondrial helicase Twinkle causes multiple mtDNA deletions and a late-onset mitochondrial disease in mice. Proc. Natl Acad. Sci. USA 102, 17687–17692 (2005).
Article ADS CAS PubMed PubMed Central Google Scholar
Christensen, A. C. et al. Dual-domain, dual-targeting organellar protein presequences in Arabidopsis can use non-AUG start codons. Plant Cell 17, 2805–28016 (2005).
Article CAS PubMed PubMed Central Google Scholar
Cheng, L., Wang, W., Yao, Y. & Sun, Q. Mitochondrial RNase H1 activity regulates R-loop homeostasis to maintain genome integrity and enable early embryogenesis in Arabidopsis. PLoS Biol. 19, e3001357 (2021).
Article CAS PubMed PubMed Central Google Scholar
Chevigny, N., Schatz-Daas, D., Lotfi, F. & Gualberto, J. M. DNA repair and the stability of the plant mitochondrial genome. Int. J. Mol. Sci. 21, (2020).
Morley, S. A., Ahmad, N. & Nielsen, B. L. Plant organelle genome replication. Plants (Basel) 8, 358 (2019).
Article CAS PubMed Google Scholar
Xiong, H.-B. et al. AtNusG, a chloroplast nucleoid protein of bacterial origin linking chloroplast transcriptional and translational machineries, is required for proper chloroplast gene expression in Arabidopsis thaliana. Nucleic Acids Res. 50, 6715–6734 (2022).
Article ADS CAS PubMed PubMed Central Google Scholar
Zhang, B. et al. Two magnesium transporters in the chloroplast inner envelope essential for thylakoid biogenesis in Arabidopsis. N. Phytol. 236, 464–478 (2022).
Article CAS Google Scholar
Gyori, B. M., Venkatachalam, G., Thiagarajan, P. S., Hsu, D. & Clement, M. V. OpenComet: an automated tool for comet assay image analysis. Redox Biol. 2, 457–465 (2014).
Article CAS PubMed PubMed Central Google Scholar

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Acknowledgements

The authors thank all the members of The Sun Lab and Professor Jie Ren (from Beijing Institute of Genomics, Chinese Academy of Sciences) for their helpful discussions and constructive suggestions. We thank Mrs. Dan Zhang and Mrs. Fang Liu (from the Center of Biomedical Analysis, Tsinghua University) for their assistance with confocal observation and Bioanalyzer analysis, respectively. We thank Mrs. Yan Yin (from the Institute of Botany, Chinese Academy of Sciences) for assisting with the measurement of chlorophyll fluorescence using FluorCam. This work was funded by grants from the National Natural Science Foundation of China (grants 32261133529 and 32170321 to Q. Sun, and 32070651 to W. Zhang). The Sun Lab is supported by the Tsinghua-Peking Center for Life Sciences. W. Zhang is supported by the China Postdoctoral Science Foundation Project (2019M660610) and the postdoctoral fellowship from Tsinghua-Peking Center for Life Sciences.

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Center for Plant Biology, School of Life Sciences, Tsinghua University, 100084, Beijing, China
Weifeng Zhang, Zhuo Yang, Wenjie Wang & Qianwen Sun
Tsinghua-Peking Center for Life Sciences, 100084, Beijing, China
Weifeng Zhang, Zhuo Yang, Wenjie Wang & Qianwen Sun

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Weifeng Zhang
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Zhuo Yang
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Wenjie Wang
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Qianwen Sun
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Contributions

Q.S. conceived the study and designed the experiments with W.Z.; Z.Y. conducted the EMS mutagenesis screen of atrnh1c suppressors; W.W. assisted W.Z. in the DEtail-seq and PFGE assays; W.Z. performed the rest of the experiments. W.Z. and Q.S. wrote the manuscript, and all authors read and approved the final manuscript.

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Correspondence to Qianwen Sun.

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Zhang, W., Yang, Z., Wang, W. et al. Primase promotes the competition between transcription and replication on the same template strand resulting in DNA damage. Nat Commun 15, 73 (2024). https://doi.org/10.1038/s41467-023-44443-0

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Received: 20 June 2023
Accepted: 13 December 2023
Published: 02 January 2024
DOI: https://doi.org/10.1038/s41467-023-44443-0

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