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

2017 ◽  
Vol 199 (17) ◽  
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.

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

Genetics ◽  
1996 ◽  
Vol 143 (1) ◽  
pp. 5-13 ◽  
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.

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

Genetics ◽  
2000 ◽  
Vol 155 (2) ◽  
pp. 487-497
Author(s):  
Steven J Sandler
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Double Mutant ◽  
Synthetic Lethality ◽  
Replication Forks ◽  
Viability Assay ◽  
Replication Restart ◽  
K 12 ◽  
Redundant Functions

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.

scholarly journals Structure-specific DNA replication-fork recognition directs helicase and replication restart activities of the PriA helicase

2018 ◽  
Vol 115 (39) ◽  
pp. E9075-E9084 ◽  
Author(s):  
Tricia A. Windgassen ◽  
Maxime Leroux ◽  
Kenneth A. Satyshur ◽  
Steven J. Sandler ◽  
James L. Keck
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Dna Helicase ◽  
Genome Replication ◽  
Duplex Dna ◽  
Winged Helix ◽  
Replication Restart ◽  
Leading Strand ◽  
Winged Helix Domain

DNA replication restart, the essential process that reinitiates prematurely terminated genome replication reactions, relies on exquisitely specific recognition of abandoned DNA replication-fork structures. The PriA DNA helicase mediates this process in bacteria through mechanisms that remain poorly defined. We report the crystal structure of a PriA/replication-fork complex, which resolves leading-strand duplex DNA bound to the protein. Interaction with PriA unpairs one end of the DNA and sequesters the 3′-most nucleotide from the nascent leading strand into a conserved protein pocket. Cross-linking studies reveal a surface on the winged-helix domain of PriA that binds to parental duplex DNA. Deleting the winged-helix domain alters PriA’s structure-specific DNA unwinding properties and impairs its activity in vivo. Our observations lead to a model in which coordinated parental-, leading-, and lagging-strand DNA binding provide PriA with the structural specificity needed to act on abandoned DNA replication forks.

Characterization of broadly pleiotropic phenotypes caused by an hfq insertion mutation in Escherichia coli K-12

1994 ◽  
Vol 13 (1) ◽  
pp. 35-49 ◽  
Author(s):  
Ho-Ching Tiffany Tsui ◽  
Hon-Chiu Eastwood Leung ◽  
Malcolm E. Winkler
Keyword(s):  
Escherichia Coli ◽  
Insertion Mutation ◽  
K 12

scholarly journals Lethal Action of Quinolones against a Temperature-Sensitive dnaB Replication Mutant of Escherichia coli

2006 ◽  
Vol 50 (1) ◽  
pp. 362-364 ◽  
Author(s):  
Xilin Zhao ◽  
Muhammad Malik ◽  
Nymph Chan ◽  
Alex Drlica-Wagner ◽  
Jian-Ying Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Dna Replication ◽  
Nalidixic Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Lethal Action ◽  
Inhibition Of Protein Synthesis

ABSTRACT Inhibition of DNA replication in an Escherichia coli dnaB-22 mutant failed to block quinolone-mediated lethality. Inhibition of protein synthesis by chloramphenicol inhibited nalidixic acid lethality and, to a lesser extent, ciprofloxacin lethality in both dnaB-22 and wild-type cells. Thus, major features of quinolone-mediated lethality do not depend on ongoing replication.

scholarly journals dnaC mutations suppress defects in DNA replication- and recombination-associated functions in priB and priC double mutants in Escherichia coli K-12

1999 ◽  
Vol 34 (1) ◽  
pp. 91-101 ◽  
Author(s):  
Steven J. Sandler ◽  
Kenneth J. Marians ◽  
Kenton H. Zavitz ◽  
Jaime Coutu ◽  
Michelle A. Parent ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Associated Functions ◽  
K 12

scholarly journals F sex factor of Escherichia coli K-12 codes for a single-stranded DNA binding protein.

1983 ◽  
Vol 80 (14) ◽  
pp. 4422-4426 ◽  
Author(s):  
A. L. Kolodkin ◽  
M. A. Capage ◽  
E. I. Golub ◽  
K. B. Low
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Single Stranded Dna ◽  
K 12

scholarly journals First-Time Isolation and Characterization of a Bacteriophage Encoding the Shiga Toxin 2c Variant, Which Is Globally Spread in Strains of Escherichia coli O157

2004 ◽  
Vol 72 (12) ◽  
pp. 7030-7039 ◽  
Author(s):  
Eckhard Strauch ◽  
Christoph Schaudinn ◽  
Lothar Beutin
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Phage Lambda ◽  
Wild Type ◽  
Chromosomal Locus ◽  
O157 Strain ◽  
Diagnostic Pcr ◽  
E Coli ◽  
Isolation And Characterization ◽  
K 12

ABSTRACT A bacteriophage encoding the Shiga toxin 2c variant (Stx2c) was isolated from the human Escherichia coli O157 strain CB2851 and shown to form lysogens on the E. coli K-12 laboratory strains C600 and MG1655. Production of Stx2c was found in the wild-type E. coli O157 strain and the K-12 lysogens and was inducible by growing bacteria in the presence of ciprofloxacin. Phage 2851 is the first reported viable bacteriophage which carries an stx 2c gene. Electron micrographs of phage 2851 showed particles with elongated hexagonal heads and long flexible tails resembling phage lambda. Sequence analysis of an 8.4-kb region flanking the stx 2c gene and other genetic elements revealed a mosaic gene structure, as found in other Stx phages. Phage 2851 showed lysis of E. coli K-12 strains lysogenic for Stx phages encoding Stx1 (H19), Stx2 (933W), Stx (7888), and Stx1c (6220) but showed superinfection immunity with phage lambda, presumably originating from the similarity of the cI repressor proteins of both phages. Apparently, phage 2851 integrates at a different chromosomal locus than Stx2 phage 933W and Stx1 phage H19 in E. coli, explaining why Stx2c is often found in combination with Stx1 or Stx2 in E. coli O157 strains. Diagnostic PCR was performed to determine gene sequences specific for phage 2851 in wild-type E. coli O157 strains producing Stx2c. The phage 2851 q and o genes were frequently detected in Stx2c-producing E. coli O157 strains, indicating that phages related to 2851 are associated with Stx2c production in strains of E. coli O157 that were isolated in different locations and time periods.

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

Genetics ◽  
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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