Multiple Genetic Pathways for Restarting DNA Replication Forks in Escherichia coli K-12

Abstract In Escherichia coli, the primosome assembly proteins, PriA, PriB, PriC, DnaT, DnaC, DnaB, and DnaG, are thought to help to restart DNA replication forks at recombinational intermediates. Redundant functions between priB and priC and synthetic lethality between priA2::kan and rep3 mutations raise the possibility that there may be multiple pathways for restarting replication forks in vivo. Herein, it is shown that priA2::kan causes synthetic lethality when placed in combination with either Δrep::kan or priC303:kan. These determinations were made using a nonselective P1 transduction-based viability assay. Two different priA2::kan suppressors (both dnaC alleles) were tested for their ability to rescue the priA-priC and priA-rep double mutant lethality. Only dnaC809,820 (and not dnaC809) could rescue the lethality in each case. Additionally, it was shown that the absence of the 3′-5′ helicase activity of both PriA and Rep is not the critical missing function that causes the synthetic lethality in the rep-priA double mutant. One model proposes that replication restart at recombinational intermediates occurs by both PriA-dependent and PriA-independent pathways. The PriA-dependent pathways require at least priA and priB or priC, and the PriA-independent pathway requires at least priC and rep. It is further hypothesized that the dnaC809 suppression of priA2::kan requires priC and rep, whereas dnaC809,820 suppression of priA2::kan does not.

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An Epistasis Analysis of recA and recN in Escherichia coli K-12

Genetics ◽

10.1534/genetics.120.303476 ◽

2020 ◽

Vol 216 (2) ◽

pp. 381-393

Author(s):

Anastasiia N. Klimova ◽

Steven J. Sandler

Keyword(s):

Escherichia Coli ◽

Double Mutant ◽

Strand Break ◽

Wild Type ◽

Epistasis Analysis ◽

Chromatid Cohesion ◽

K 12 ◽

Structural Maintenance Of Chromosomes ◽

Sos Protein

RecA is essential for double-strand-break repair (DSBR) and the SOS response in Escherichia coli K-12. RecN is an SOS protein and a member of the Structural Maintenance of Chromosomes family of proteins thought to play a role in sister chromatid cohesion/interactions during DSBR. Previous studies have shown that a plasmid-encoded recA4190 (Q300R) mutant had a phenotype similar to ∆recN (mitomycin C sensitive and UV resistant). It was hypothesized that RecN and RecA physically interact, and that recA4190 specifically eliminated this interaction. To test this model, an epistasis analysis between recA4190 and ∆recN was performed in wild-type and recBC sbcBC cells. To do this, recA4190 was first transferred to the chromosome. As single mutants, recA4190 and ∆recN were Rec+ as measured by transductional recombination, but were 3-fold and 10-fold decreased in their ability to do I-SceI-induced DSBR, respectively. In both cases, the double mutant had an additive phenotype relative to either single mutant. In the recBC sbcBC background, recA4190 and ∆recN cells were very UVS (sensitive), Rec−, had high basal levels of SOS expression and an altered distribution of RecA-GFP structures. In all cases, the double mutant had additive phenotypes. These data suggest that recA4190 (Q300R) and ∆recN remove functions in genetically distinct pathways important for DNA repair, and that RecA Q300 was not important for an interaction between RecN and RecA in vivo. recA4190 (Q300R) revealed modest phenotypes in a wild-type background and dramatic phenotypes in a recBC sbcBC strain, reflecting greater stringency of RecA’s role in that background.

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Anatomy of a twin DNA replication factory

Biochemical Society Transactions ◽

10.1042/bst20200640 ◽

2020 ◽

Vol 48 (6) ◽

pp. 2769-2778

Author(s):

Huilin Li ◽

Nina Y. Yao ◽

Michael E. O'Donnell

Keyword(s):

Escherichia Coli ◽

Saccharomyces Cerevisiae ◽

Bacillus Subtilis ◽

Dna Replication ◽

In Vivo Studies ◽

Replication Forks ◽

Structural Studies ◽

Replication Factory ◽

Higher Eukaryotes

The replication of DNA in chromosomes is initiated at sequences called origins at which two replisome machines are assembled at replication forks that move in opposite directions. Interestingly, in vivo studies observe that the two replication forks remain fastened together, often referred to as a replication factory. Replication factories containing two replisomes are well documented in cellular studies of bacteria (Escherichia coli and Bacillus subtilis) and the eukaryote, Saccharomyces cerevisiae. This basic twin replisome factory architecture may also be preserved in higher eukaryotes. Despite many years of documenting the existence of replication factories, the molecular details of how the two replisome machines are tethered together has been completely unknown in any organism. Recent structural studies shed new light on the architecture of a eukaryote replisome factory, which brings with it a new twist on how a replication factory may function.

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A priA Mutant Expressed in Two Pieces Has Almost Full Activity in Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.00267-17 ◽

2017 ◽

Vol 199 (17) ◽

Cited By ~ 2

Author(s):

Maxime Leroux ◽

Niketa Jani ◽

Steven J. Sandler

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Dna Binding ◽

Insertion Mutation ◽

Wild Type ◽

Poor Growth ◽

Winged Helix ◽

Replication Restart ◽

K 12 ◽

Winged Helix Domain

ABSTRACT The ability to restart broken DNA replication forks is essential across all domains of life. In Escherichia coli, the priA, priB, priC, and dnaT genes encode the replication restart proteins (RRPs) to accomplish this task. PriA plays a critical role in replication restart such that its absence reveals a dramatic phenotype: poor growth, high basal levels of SOS expression, poorly partitioned nucleoids (Par−), UV sensitivity, and recombination deficiency (Rec−). PriA has 733 amino acids, and its structure is composed of six domains that enable it to bind to DNA replication fork-like structures, remodel the strands of DNA, interact with SSB (single-stranded DNA binding protein), PriB, and DnaT, and display ATPase, helicase, and translocase activities. We have characterized a new priA mutation called priA316::cat. It is a composite mutation involving an insertion that truncates the protein within the winged-helix domain (at the 154th codon) and an ACG (Thr)-to-ATG (Met) mutation that allows reinitiation of translation at the 157th codon such that PriA is expressed in two pieces. priA316::cat phenotypes are like those of the wild type for growth, recombination, and UV resistance, revealing only a slightly increased level of SOS expression and defects in nucleoid partitioning in the mutant. Both parts of PriA are required for activity, and the N-terminal fragment can be optimized to yield wild-type activity. A deletion of the lon protease suppresses priA316::cat phenotypes. We hypothesize the two parts of PriA form a complex that supplies most of the PriA activity needed in the cell. IMPORTANCE PriA is a highly conserved multifunctional protein that plays a crucial role in the essential process of replication restart. Here we characterize an insertion mutation of priA with an intragenic suppressor such that it is now made in two parts. These two pieces split the winged-helix domain to separate the N-terminal 3′ DNA-binding domain from the C-terminal domain of PriA. It is hypothesized that the two pieces form a complex that is capable of almost wild type priA function. The composite mutation leads to a moderate level of SOS expression and defects in partitioning of the chromosomes. Full function is restored by deletion of lon, suggesting that stability of this complex may be a reason for the partial phenotypes seen.

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DNA Helicase-SSB Interactions Critical to the Regression and Restart of Stalled DNA Replication Forks in Escherichia coli

Genes ◽

10.3390/genes11050471 ◽

2020 ◽

Vol 11 (5) ◽

pp. 471 ◽

Cited By ~ 1

Author(s):

Piero R. Bianco

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Dna Helicase ◽

Dependent Manner ◽

Replication Forks ◽

Replication Restart ◽

Helicase Dnab ◽

Fork Regression ◽

Ob Fold ◽

Dna Replication Restart

In Escherichia coli, DNA replication forks stall on average once per cell cycle. When this occurs, replisome components disengage from the DNA, exposing an intact, or nearly intact fork. Consequently, the fork structure must be regressed away from the initial impediment so that repair can occur. Regression is catalyzed by the powerful, monomeric DNA helicase, RecG. During this reaction, the enzyme couples unwinding of fork arms to rewinding of duplex DNA resulting in the formation of a Holliday junction. RecG works against large opposing forces enabling it to clear the fork of bound proteins. Following subsequent processing of the extruded junction, the PriA helicase mediates reloading of the replicative helicase DnaB leading to the resumption of DNA replication. The single-strand binding protein (SSB) plays a key role in mediating PriA and RecG functions at forks. It binds to each enzyme via linker/OB-fold interactions and controls helicase-fork loading sites in a substrate-dependent manner that involves helicase remodeling. Finally, it is displaced by RecG during fork regression. The intimate and dynamic SSB-helicase interactions play key roles in ensuring fork regression and DNA replication restart.

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Differential Suppression of priA2::kan Phenotypes in Escherichia coli K-12 by Mutations in priA, lexA, and dnaC

Genetics ◽

10.1093/genetics/143.1.5 ◽

1996 ◽

Vol 143 (1) ◽

pp. 5-13 ◽

Cited By ~ 21

Author(s):

Steven J Sandler ◽

Hardeep S Samra ◽

Alvin J Clark

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Mutant Allele ◽

Wild Type ◽

Suppressor Mutations ◽

Dnab Helicase ◽

K 12 ◽

Extragenic Suppressors ◽

Assembly Activity

Abstract First identified as an essential component of the ϕX174 in vitro DNA replication system, PriA has ATPase, helicase, translocase, and primosome-assembly activities. priA1::kan strains of Escherichia coli are sensitive to UV irradiation, deficient in homologous recombination following transduction, and filamentous. priA2::kan strains have eightfold higher levels of uninduced SOS expression than wild type. We show that (1) priA1::kan strains have eightfold higher levels of uninduced SOS expression, (2) priA2::kan strains are UVS and Rec−, (3) lexA3 suppresses the high basal levels of SOS expression of a priA2::kan strain, and (4) plasmid-encoded priA300 (K230R), a mutant allele retaining only the primosome-assembly activity of priA+, restores both UVR and Rec+ phenotypes to a priA2::kan strain. Finally, we have isolated 17 independent UVR Rec+ revertants of priA2::kan strains that carry extragenic suppressors. All 17 map in the C-terminal half of the dnaC gene. DnaC loads the DnaB helicase onto DNA as a prelude for primosome assembly and DNA replication. We conclude that priA's primosome-assembly activity is essential for DNA repair and recombination and that the dnaC suppressor mutations allow these processes to occur in the absence of priA.

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Specific in vivo thiol-labeling of the FhuA outer membrane ferrichrome transport protein of Escherichia coli K-12: evidence for a disulfide bridge in the predicted gating loop

FEMS Microbiology Letters ◽

10.1111/j.1574-6968.1997.tb12590.x ◽

2006 ◽

Vol 153 (2) ◽

pp. 311-319

Author(s):

Christoph Bös ◽

Volkmar Braun

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Disulfide Bridge ◽

Transport Protein ◽

K 12

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Single-molecule binding characterization of primosomal protein PriA involved in replication restart

Physical Chemistry Chemical Physics ◽

10.1039/d1cp00638j ◽

2021 ◽

Author(s):

Tzu-Yu Lee ◽

Yi-Ching Li ◽

Min-Guan Lin ◽

Chwan-Deng Hsiao ◽

Hung-Wen Li

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Single Molecule ◽

Replication Forks ◽

Bacterial Survival ◽

Dna Damages ◽

Replication Restart

DNA damages lead to stalled or collapsed replication forks. Replication restart primosomes re-initiate DNA synthesis at these stalled or collapsed DNA replication forks, which is important for bacterial survival. Primosomal...

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