The replisome uses mRNA as a primer after colliding with RNA polymerase

110

152

DNA replication fork movement is impeded by collisions with transcription elongation complexes (TEC). We propose that a critical function of transcription termination factors is to prevent TEC from blocking DNA replication and inducing replication fork arrest, one consequence of which is DNA double-strand breaks. We show that inhibition of Rho-dependent transcription termination by bicyclomycin in Escherichia coli induced double-strand breaks. Cells deleted for Rho-cofactors nusA and nusG were hypersensitive to bicyclomycin, and had extensive chromosome fragmentation even in the absence of the drug. An RNA polymerase mutation that destabilizes TEC (rpoB*35) increased bicyclomycin resistance >40-fold. Double-strand break formation depended on DNA replication, and can be explained by replication fork collapse. Deleting recombination genes required for replication fork repair (recB and ruvC) increased sensitivity to bicyclomycin, as did loss of the replication fork reloading helicases rep and priA. We propose that Rho responds to a translocating replisome by releasing obstructing TEC.DNA polymerase/RNA polymerase collisions | pulsed-field gel electrophoresis | SOS D NA replication forks translocate 20 to 30 times faster (∼600 nt/s) than the rate of RNA polymerase (RNAP) elongation (∼20 nt/s) (1-3). This finding raises the possibility of repeated collisions between replication forks and transcription elongation complexes (TEC), even when DNA and RNA synthesis are codirectional. Collisions between replication forks and TEC can generate fork collapse and chromosomal double-strand breaks (DSBs) (4, 5). Mechanisms have evolved, therefore, to reduce collisions and to repair collapsed forks. Fork repair is promoted by multiple pathways that involve a variety of DNA repair functions (6). Recombination can restart replisomes blocked by arrested TEC (7,8). Boubakri et al. (9) have demonstrated that DNA helicases Rep, DinG, and UvrD resolve head-on collisions between TEC in rrn operons and replisomes. A recent report describes the role of transcription elongation factors DksA and GreA in preventing conflicts between replication and transcription (7, 10). In vitro, replication forks that collapse after colliding with a codirectional TEC can restart, using an R-loop (RNA-DNA hybrid) from a transcription bubble as primer (11), although it is not known if this occurs in vivo.Transcription termination isolates transcription units and prevents inappropriate expression of downstream genes. Termination in Escherichia coli is largely mediated by Rho, an RNAdependent ATPase that terminates transcription promoter-distal to unstructured and untranslated RNA (12, 13). The RNA/DNA helicase activity of Rho promotes termination by unwinding RNA/DNA hybrid in the RNAP transcription bubble (14). Inhibition of Rho with bicyclomycin (BCM) or deletion of the Rho accessory factors, NusA and NusG, massively disregulate gene expression in E. coli (15). Surprisingly, efficient transcription termination is only required to suppress expression of cryp...

Section: Discussionmentioning

confidence: 86%

mentioning

confidence: 99%

Transcription termination maintains chromosome integrity

Washburn

Gottesman²

2010

110

152

“…S2A). A truncated replication machinery potentially restarts replication from an R-loop, given that elegant in vitro experiments demonstrated that replication can restart from a purified E. coli replisome-RNAP complex, and that the replisome uses mRNA as a primer to reinitiate leading-strand synthesis after displacing a codirectional RNAP from DNA (56). One can speculate that RNAPI is no longer associated with the R-loop, a scenario that facilitates TIR without the need for factors that drive the displacement of RNAPs being head-on to a replisome (57).…”

Section: Which Factors and Mechanism Would Participate In Transcription-mentioning

confidence: 99%

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

Stuckey

García‐Rodríguez

Aguilera

et al. 2015

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...

“…If forks pause at such barriers and lose function, then replisome reloading, often via blocked fork processing by recombination enzymes, is required to resume genome duplication (4), an error-prone process associated with gross chromosomal rearrangements (5)(6)(7). However, pausing of replisomes does not necessarily lead to fork breakdown, because paused replisomes can continue duplication upon removal or bypass of the block (8)(9)(10)(11). The balance between resumption of replication versus breakdown of paused forks is therefore a critical factor in the maintenance of genome stability.…”

mentioning

confidence: 99%

Protein–DNA complexes are the primary sources of replication fork pausing in Escherichia coli

Gupta

Guy

Yeeles

et al. 2013

105

Replication fork pausing drives genome instability, because any loss of paused replisome activity creates a requirement for reloading of the replication machinery, a potentially mutagenic process. Despite this importance, the relative contributions to fork pausing of different replicative barriers remain unknown. We show here that Deinococcus radiodurans RecD2 helicase inactivates Escherichia coli replisomes that are paused but still functional in vitro, preventing continued fork movement upon barrier removal or bypass, but does not inactivate elongating forks. Using RecD2 to probe replisome pausing in vivo, we demonstrate that most pausing events do not lead to replisome inactivation, that transcription complexes are the primary sources of this pausing, and that an accessory replicative helicase is critical for minimizing the frequency and/or duration of replisome pauses. These findings reveal the hidden potential for replisome inactivation, and hence genome instability, inside cells. They also demonstrate that efficient chromosome duplication requires mechanisms that aid resumption of replication by paused replisomes, especially those halted by protein-DNA barriers such as transcription complexes.DNA repair | genome stability | Rep | RNA polymerase | recombination