Primase excites the competition of templating transcription and lagging replication to boost DNA damage. Primase excites the competition of templating transcription and lagging replication to boost DNA damage

Head-on transcription-replication conflicts (HO-TRCs) cause R-loops and DNA damage. We realized that HO-TRCs are the natural competition for templating transcription and lagging replication in the same DNA strand (termed TemLag competition), however, the regulatory mechanisms behind are 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 identified that the chloroplast-localized primase ATH exacerbates TemLag competition in the atrnh1c mutant. A mutation in ATH weakens the binding affinity of DNA, thus slowing down DNA replication and relieving TemLag competition. Overexpression of ATH boosts TemLag competition and intensifies DNA damage. Interestingly, strand-specific DNA damage sequencing reveals that TemLag competition induces single-strand DNA damage of the competitive strand at the end of transcription. These results illustrated a potentially conserved mechanism among organisms, of which the primase antagonizes R-loop clearance machinery to exaggerate TemLag competition and genome instability. Overall design: Detail-seq performed using chloroplasts embedded in low-melting-point agarose. ssDRIP performed with S9.6 antibody in atrnh1c mutant. ATH ChIP performed with GFP antibody using ATH-GFP tagged transgenic plants, and Col-0 with no tag as control.