Dataset Information

SSB binds to the RecG and PriA helicases in vivo in the absence of DNA.

ABSTRACT: The E. coli single-stranded DNA-binding protein (SSB) binds to the fork DNA helicases RecG and PriA in vitro. Typically for binding to occur, 1.3 m ammonium sulfate must be present, bringing into question the validity of these results as these are nonphysiological conditions. To determine whether SSB can bind to these helicases, we examined binding in vivo. First, using fluorescence microscopy, we show that SSB localizes PriA and RecG to the vicinity of the inner membrane in the absence of DNA damage. Localization requires that SSB be in excess over the DNA helicases and the SSB C-terminus and both PriA and RecG be present. Second, using the purification of tagged complexes, our results show that SSB binds to PriA and RecG in vivo, in the absence of DNA. We propose that this may be the 'storage form' of RecG and PriA. We further propose that when forks stall, RecG and PriA are targeted to the fork by SSB, which, by virtue of its high affinity for single-stranded DNA, allows these helicases to outcompete other proteins. This ensures their actions in the early stages of the rescue of stalled replication forks.

SUBMITTER: Yu C

PROVIDER: S-EPMC4738028 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

SSB binds to the RecG and PriA helicases in vivo in the absence of DNA.

Yu Cong C Tan Hui Yin HY Choi Meerim M Stanenas Adam J AJ Byrd Alicia K AK D Raney Kevin K Cohan Christopher S CS Bianco Piero R PR

Genes to cells : devoted to molecular & cellular mechanisms 20160113 2

The E. coli single-stranded DNA-binding protein (SSB) binds to the fork DNA helicases RecG and PriA in vitro. Typically for binding to occur, 1.3 m ammonium sulfate must be present, bringing into question the validity of these results as these are nonphysiological conditions. To determine whether SSB can bind to these helicases, we examined binding in vivo. First, using fluorescence microscopy, we show that SSB localizes PriA and RecG to the vicinity of the inner membrane in the absence of DNA d ...[more]

PMID: 26766785

Similar Datasets

Project description:Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par-). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par- and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB.IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

Dataset Information

SSB binds to the RecG and PriA helicases in vivo in the absence of DNA.

Publications

SSB binds to the RecG and PriA helicases in vivo in the absence of DNA.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets