Regression supports two mechanisms of fork processing in phage T4

Abstract

Replication forks routinely encounter damaged DNA and tightly bound proteins, leading to fork stalling and inactivation. To complete DNA synthesis, it is necessary to remove fork-blocking lesions and reactivate stalled fork structures, which can occur by multiple mechanisms. To study the mechanisms of stalled fork reactivation, we used a model fork intermediate, the origin fork, which is formed during replication from the bacteriophage T4 origin, ori(34). The origin fork accumulates within the T4 chromosome in a site-specific manner without the need for replication inhibitors or DNA damage. We report here that the origin fork is processed in vivo to generate a regressed fork structure. Furthermore, origin fork regression supports two mechanisms of fork resolution that can potentially lead to fork reactivation. Fork regression generates both a site-specific double-stranded end (DSE) and a Holliday junction. Each of these DNA elements serves as a target for processing by the T4 ATPase/exonuclease complex [gene product (gp) 46/47] and Holliday junction-cleaving enzyme (EndoVII), respectively. In the absence of both gp46 and EndoVII, regressed origin forks are stabilized and persist throughout infection. In the presence of EndoVII, but not gp46, there is significantly less regressed origin fork accumulation apparently due to cleavage of the regressed fork Holliday junction. In the presence of gp46, but not EndoVII, regressed origin fork DSEs are processed by degradation of the DSE and a pathway that includes recombination proteins. Although both mechanisms can occur independently, they may normally function together as a single fork reactivation pathway.2D gel electrophoresis ͉ DNA replication ͉ recombination ͉ restart R eplication forks routinely encounter various lesions, ranging from damaged nucleotide residues to covalent protein-DNA complexes. The consequences of these encounters depend on the type and position of the lesion (1). Although some lesions result in fork breakage, many result in fork stalling and replisome inactivation. Presumably, replisome disassembly is important for the repair of the original DNA lesion. However, it is then necessary to reactivate stalled forks to complete replication and maintain genome stability. Therefore, it is critical to understand the mechanisms of stalled fork resolution and reactivation.To identify the pathways of stalled fork reactivation, we have used a model fork intermediate that is formed during replication from bacteriophage T4 origin, ori(34). Replication from ori(34) occurs bidirectionally through a two-step process (Fig. 1) (2). A middle-mode promoter and DNA unwinding element promote the formation of an R loop that initiates the first replication fork (3). The mechanism of T4 replication from an R loop has been analyzed in vitro (4). Initially, the branch structure of the R loop supports the binding of the replicative helicase loader [gene product (gp) 59], which promotes removal of the ssDNA-binding protein (gp32) in favor of the replicative helicase (gp41). ...

Fusion of nearby inverted repeats by a replication-based mechanism leads to formation of dicentric and acentric chromosomes that cause genome instability in budding yeast

“…7). This mechanism bears a similarity to mechanisms proposed from studies in bacteria (Goldfless et al 2006;Long and Kreuzer 2008;Atkinson and McGlynn 2009). In this model, a stalled fork fails to be rescued by error-free pathways (the Rad51, Rad18-dependent hemicatenane pathway, for example) (see Fig.…”

SRS2 and SGS1 prevent chromosomal breaks and stabilize triplet repeats by restraining recombination

“…3b). This conical shape contains reversed replication forks whose double-stranded ends have been partially resected by the gp46 nuclease 51 .…”

Diversity and evolution of chromatin proteins encoded by DNA viruses

“…ends of two separate chromosomes), with each ATPase head contacting one of the units that are being bridged [100]. This bridging function of Rad50 appears to be critical in bringing chromosomes together for non-homologous end joining and a comparable role has been reported for SbcC and gp46 in processing dsDNA ends for processing during recombination [101, 102]. Our analysis shows that the majority of viruses (70%) with SbcC/gp46-like proteins possess linear chromosomes.…”