Role for RNA:DNA hybrids in origin-independent replication priming in a eukaryotic system

Abstract

DNA replication initiates at defined replication origins along eukaryotic chromosomes, ensuring complete genome duplication within a single S-phase. A key feature of replication origins is their ability to control the onset of DNA synthesis mediated by DNA polymerase-α and its intrinsic RNA primase activity. Here, we describe a novel origin-independent replication process that is mediated by transcription. RNA polymerase I transcription constraints lead to persistent RNA:DNA hybrids (R-loops) that prime replication in the ribosomal DNA locus. Our results suggest that eukaryotic genomes have developed tools to prevent R-loop-mediated replication events that potentially contribute to copy number variation, particularly relevant to carcinogenesis.D uring transcription, DNA acts as a template for the synthesis of the nascent RNA. RNA synthesis is accompanied by the generation of positive and negative DNA supercoiling in front of and behind the transcription machinery, respectively (1). Unwinding of the DNA double helix by negative supercoiling may allow the RNA to hybridize to its DNA template behind the elongating RNA polymerase, leading to R-loops (2). Other elements that could potentiate R-loop accumulation include RNA: DNA hybrid-facilitating DNA sequences, such as G-quadruplex structures (3) or nicks in the nontemplate DNA strand (4).Eukaryotic cells need to control R-loop formation to avoid replication impairment, genome instability, and life span shortening mediated by such intermediates (5-10; reviewed in ref. 11). To do so, cells catalyze the relaxation of supercoiled DNA by type I topoisomerases (12-15), thus preventing replication fork reversal (16), DNA overwinding with the potential to block replication fork progression (17), DNA unwinding (18), or R-loop-mediated blocks of ribosomal RNA synthesis (19). Other enzymatic activities involved in R-loop processing include ribonuclease H (RNase H) activities, DNA-RNA helicases, such as Sen1/senataxin (20, 21), or Ataxin-2 RNA-binding protein Pbp1 (10). The ribonuclease activity of Saccharomyces cerevisiae RNases H1 and H2 specifically cleaves the RNA moiety of the RNA:DNA hybrid structure (22), whereas RNase H2 and topoisomerase 1 (Top1) can also process ribonucleotides in duplex DNA (23,24).Notably, R-loops are required to initiate mitochondrial DNA replication (25) and pioneering studies connected R-loops to origin-independent replication in prokaryotic systems (26,27). For example, DnaA-dependent initiation of DNA replication at the Escherichia coli oriC replication origin can be overcome in the absence of RNase H1 (28, 29). As a consequence, rnhA mutants can survive complete inactivation of oriC by transcriptiondependent activation of so-called oriK sites (30, 31), although candidate oriK sites have been identified only recently (32). Additional evidence for R-loop-primed replication was given by the observation that rnhA mutants are prone to an increase in mutation and DNA amplification events if origin activity is suppressed. These events required remov...

RNase H enables efficient repair of R-loop induced DNA damage

“…These alleles were particularly intriguing because previous studies have shown that the majority of Rad52-GFP foci seen in RNase H mutants co-localize with the nucleolus. This suggested that the lethal events seen in rnh1D rnh201D top1D cells were rDNA-specific (Stuckey et al, 2015). Consistent with these findings, we observed efficient repair of a DSB on chromosome V by BIR and NHEJ in top1D and rnh1D rnh201D cells.…”

“…Consistent with the notion that persistent DNA damage uniquely affects the double mutants, the growth of the double mutant, but not either of the single mutants, was dramatically impaired by the deletion of RAD52 ( Figure 1B). Previous characterization of the double mutant also reported elevated foci and Rad52-dependent growth (Stuckey et al, 2015;Lazzaro et al, 2012). Thus, by measures of Rad52-GFP foci and Rad52-dependent growth, cells lacking RNase H1 and H2 had a larger fraction of persistent R-loop induced damage than wild-type cells or cells lacking only one of the RNases H. This persistent damage could have arisen from increased R-loop induced damage and/or an inability to efficiently repair that damage.…”

“…A potential synergistic relationship between Top1 and the RNases H came from the observation that while cells with either the top1D mutation or the rnh1D rnh201D mutations are viable, the top1D rnh1D rnh201D mutant is inviable (El Hage et al, 2010). Furthermore, treatment of rnh1D rnh201D cells with the Top1 inhibitor camptothecin led to increased Rad52-GFP foci that co-localized with the nucleolus (Stuckey et al, 2015). Encouraged by these results, we used the auxininducible degron (AID) system to create a conditional TOP1-AID allele in wild-type, the two single RNase H mutants, and the double RNase H mutant.…”

“…Consistent with phenotypes of increased genomic instability, rnh1D rnh201D mutants display an increase in Rad52-GFP foci. A large fraction of these foci appear to co-localize with the nucleolus and form in a window between late S and mid-M (Stuckey et al, 2015). Here, by monitoring the persistence of Rad52 foci across the cell cycle in RNase H mutants, we implicate DNA:RNA hybrids in the disruption of DNA repair.…”

“…Accordingly, almost 50% of all R-loops map to the rDNA (Wahba et al, 2016). R-loops found at the rDNA are associated with increased rates of recombination (Wahba et al, 2011(Wahba et al, , 2013, RNA polymerase pileups (El Hage et al, 2010), and stalled replication forks (Stuckey et al, 2015).…”