Processing of stalled replication forks in Bacillus subtilis

Abstract

Accurate DNA replication and transcription elongation are crucial for preventing the accumulation of unreplicated DNA and genomic instability. Cells have evolved multiple mechanisms to deal with impaired replication fork progression, challenged by both intrinsic and extrinsic impediments. The bacterium Bacillus subtilis, which adopts multiple forms of differentiation and development, serves as an excellent model system for studying the pathways required to cope with replication stress to preserve genomic stability. This review focuses on the genetics, single molecule choreography, and biochemical properties of the proteins that act to circumvent the replicative arrest allowing the resumption of DNA synthesis. The RecA recombinase, its mediators (RecO, RecR, and RadA/Sms) and modulators (RecF, RecX, RarA, RecU, RecD2, and PcrA), repair licensing (DisA), fork remodelers (RuvAB, RecG, RecD2, RadA/Sms, and PriA), Holliday junction resolvase (RecU), nucleases (RnhC and DinG), and translesion synthesis DNA polymerases (PolY1 and PolY2) are key functions required to overcome a replication stress, provided that the fork does not collapse.

Keywords: DNA damage tolerance; RNA polymerase hub; RecA hub; SsbA hub; fork reversal; replisome disassembly; template switching.

Figure 1.

Potential replication stress response mechanisms. A replicative DNAP cannot accommodate a damaged template base (represented by a red dot) and transiently stalls. Replication may proceed via error-free (template switching, fork remodeling) or error-prone DDT pathways to allow replication to resume (A)–(E) or restart (F)–(I). The replicative DNAP may skip the lesion, and upon reloading of the primosomal complex and repriming, DNA synthesis continues. The resulting lesion-containing gap left behind is filled by template switching, mainly via a RecA-dependent mechanism (A)–(C). The replicative DNAP may be replaced by a specialized TLS DNAP that often incorporates an erroneous nucleotide opposite the damaged template, leading to mutagenesis (denoted by x) (D) and (E). Enzyme-catalyzed reversal of the stalled fork by annealing the nascent strands occurs, with the nascent leading-strand extended (F). The fork can be restored by regressing the reversed fork, or the nascent lagging-strand is removed to generate a 3′-fork DNA for replication restart (G)–(I). Alternatively, the nascent lagging-strand is removed to generate a 3′-fork DNA for replication restart (H) and (I).

Figure 2.

Protein–protein interaction network in B. subtilis. RNAP, RecA, and SsbA are protein–protein interaction hubs that may connect several proteins involved in the processing of stalled replication forks. Solid lines show protein–protein interactions proven by pull-downs, bacterial two-hybrid system, and/or confirmed by biochemical or biophysical experiments. The dotted lines show suggested interactions.

Figure 3.

Proposed model for remodeling stalled forks in B. subtilis. (A) and (G) When a replisome encounters a lesion in the template strand, it stalls and disassembles. SsbA bound to the resulting lesion-containing gap on the template leading-strand [termed here forked-Lead (A)] or on the template lagging-strand [termed here forked-Lag (G)] inhibits RecA loading. Mediators such as RecO (or RecO–RecR, not depicted), or RadA/Sms, displace SsbA, and interact with and recruit RecA, which then binds onto the lesion-containing gap on the template strand. DisA scans the genome, searching for branched intermediates, and pauses. DisA interacts with and inhibits the ATPase of RecA, and this indirectly avoids filament growth and SOS induction. (B) and (H) RecA bound to the template strand interacts with and loads the RadA/Sms helicase on the nascent-lagging-strand, with RadA/Sms unwinding it. (C) and (I) Spontaneous remodeling (or fork remodeler-mediated) places the deleterious lesion on duplex DNA for its removal by specialized pathways. Finally, PriA, which recognizes a 3′-fork DNA, recruits other preprimosomal proteins (DnaD–DnaD–DnaI) to load the DnaC helicase for replication restart. (D) and (J). Alternatively, the RecG remodeler converts forked-Lead (A) into a HJ DNA with a nascent lagging-strand longer than the leading-strand (termed here HJ-Lag DNA) (D), or the forked-Lag (G) into a HJ DNA with a nascent leading-strand longer than the lagging-strand (termed here HJ-Lead DNA) (J). DisA bound to these HJ structures limits RecG or RuvAB mediated branch migration, and RuvAB–RecU-mediated HJ cleavage. RadA/Sms bound itself (E), or been recruited by RecA bound to HJ-Lead DNA (K), unwinds the nascent lagging-strand to yield a 3′-fork DNA. Then, PriA bound to the 3′-fork DNA substrate recruits other preprimosomal components to reinitiate DNA replication (F) and (L). (E) RecA, with the help of its accessory proteins (RecO and SsbA) or DisA may limit RadA/Sms loading at the 5′-tailed HJ-Lag DNA to facilitate that DNA synthesis occurs by the extension of the nascent leading-strand using the nascent lagging-strand as a template to bypass the deleterious lesion. (K) DisA and SsbA may regulate RadA/Sms recruitment by RecA to HJ-Lead DNA.

Figure 4.

Cartoon showing how B. subtilis proteins may contribute to resolve RTCs. Here, a CD RTC is illustrated. A replisome clashes with multiple RNAPs transcribing highly expressed genes (i.e. rRNA genes), or the RNAP finds a DNA lesion on the template strand (red circle), and transcription is halted. Upon that, the stalled fork has a tendency to reverse and RNAP to backtrack, and this causes topological constrains that facilitate R-loop formation. RecA (purple circles) may bind to the ssDNA region in the regressed fork or in the R-loop. RNase J1 (green Pac-Man) or FenA (yellow Pac-Man) degrades the mRNA. RNAP and RecA, acting as hubs, interact with and recruit PcrA (orange drop), RnhC (black Pac-Man), or DinG (grey Pac-Man) at the trafficking conflict. PcrA displaces and RnhC degrades the RNA strand of the R-loop. PcrA or YwqA facilitates RNAP backtracking, Mfd or HelD facilitates RNAP removal and DinG degrades the exposed 3′-end of the mRNA to facilitate transcription reinitiation upon removal of the lesions. RecA bound to the reversed fork may protect it from degradation. Although not depicted here, topoisomerases may also play a role at RTC sites.