Mutant single-strand binding protein of Escherichia coli: genetic and physiological characterization

Glassberg, Jeffrey; Meyer, Ralph R.; Kornberg, Arthur

doi:10.1128/jb.140.1.14-19.1979

Cited by 180 publications

(42 citation statements)

References 28 publications

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“…The interaction of many proteins with the SSB C-terminal tail is disrupted by mutation of the penultimate amino acid Pro 176 to Ser (33). Strains carrying this mutation (ssb-113, originally lexC113) are temperature sensitive and quite compromised (34,35). To investigate this question, we constructed W3110 topB-ecgfp S mCherry-dnaN ssb-113 (CL281) and compared Topo III and ␤ localization with that in the wild-type strain (CL109) at 30°C in minimal medium (Fig.…”

Section: Resultsmentioning

confidence: 99%

Topoisomerase III Acts at the Replication Fork To Remove Precatenanes

et al. 2019

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The role of DNA topoisomerase III (Topo III) in bacterial cells has proven elusive. Whereas eukaryotic Top III␣ homologs are clearly involved with homologs of the bacterial DNA helicase RecQ in unraveling double Holliday junctions, preventing crossover exchange of genetic information at unscheduled recombination intermediates, and Top III␤ homologs have been shown to be involved in regulation of various mRNAs involved in neuronal function, there is little evidence for similar reactions in bacteria. Instead, most data point to Topo III playing a role supplemental to that of topoisomerase IV in unlinking daughter chromosomes during DNA replication. In support of this model, we show that Escherichia coli Topo III associates with the replication fork in vivo (likely via interactions with the single-stranded DNAbinding protein and the ␤ clamp-loading DnaX complex of the DNA polymerase III holoenzyme), that the DnaX complex stimulates the ability of Topo III to unlink both catenated and precatenated DNA rings, and that ΔtopB cells show delayed and disorganized nucleoid segregation compared to that of wild-type cells. These data argue that Topo III normally assists topoisomerase IV in chromosome decatenation by removing excess positive topological linkages at or near the replication fork as they are converted into precatenanes. IMPORTANCE Topological entanglement between daughter chromosomes has to be reduced to exactly zero every time an E. coli cell divides. The enzymatic agents that accomplish this task are the topoisomerases. E. coli possesses four topoisomerases. It has been thought that topoisomerase IV is primarily responsible for unlinking the daughter chromosomes during DNA replication. We show here that topoisomerase III also plays a role in this process and is specifically localized to the replisome, the multiprotein machine that duplicates the cell's genome, in order to do so.

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Section: Resultsmentioning

confidence: 99%

Topoisomerase III Acts at the Replication Fork To Remove Precatenanes

et al. 2019

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“…The temperature defect of SSB‐113 in in vitro reactions using Pol III holoenzyme is explained further in the Discussion. Alternatively, the χ–SSB contact is involved in the repair‐ and recombination‐defective phenotypes of SSB‐113 that occur at all temperatures examined (Glassberg et al ., 1979; Vales et al ., 1980; Golub and Low, 1983; Lieberman and Witkin, 1983; Whittier and Chase, 1983; Laine and Meyer, 1992).…”

Section: Resultsmentioning

confidence: 99%

Devoted to the lagging strand_the chi subunit of DNA polymerase III holoenzyme contacts SSB to promote processive elongation and sliding clamp assembly

et al. 1998

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Escherichia coli DNA polymerase III holoenzyme contains 10 different subunits which assort into three functional components: a core catalytic unit containing DNA polymerase activity, the β sliding clamp that encircles DNA for processive replication, and a multisubunit clamp loader apparatus called γ complex that uses ATP to assemble the β clamp onto DNA. We examine here the function of the χ subunit of the γ complex clamp loader. Omission of χ from the holoenzyme prevents contact with single-stranded DNA-binding protein (SSB) and lowers the efficiency of clamp loading and chain elongation under conditions of elevated salt. We also show that the product of a classic point mutant of SSB, SSB-113, lacks strong affinity for χ and is defective in promoting clamp loading and processive replication at elevated ionic strength. SSB-113 carries a single amino acid replacement at the penultimate residue of the C-terminus, indicating the C-terminus as a site of interaction with χ. Indeed, a peptide of the 15 C-terminal residues of SSB is sufficient to bind to χ. These results establish a role for the χ subunit in contacting SSB, thus enhancing the clamp loading and processivity of synthesis of the holoenzyme, presumably by helping to localize the holoenzyme to sites of SSB-coated ssDNA.

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“…In vivo studies have shown that, in addition to their well known roles in DNA replication, E.coli SSB protein (Glassberg et al, 1979), T4 gene 32 protein (Tomizawa et al, 1966;Berger et al, 1969;Mosig et al, 1979) and T7 gene 2.5 protein (Araki and Ogawa, 1981) are also involved in recombination. However, the multiple reactions in which these proteins participate have made it difficult to dissect their contributions to recombination and replication since they may serve identical roles in both processes.…”

Section: Discussionmentioning

confidence: 99%

“…However, other proteins can mediate some of the subsequent steps in recombination equally as well as the RecA-type protein. For example, the single-stranded DNA (ssDNA) binding proteins, of which the E.coli SSB protein and the T4 gene 32 protein are examples, stimulate the annealing of complementary DNA strands (Chase and Williams, 1986), and in vivo studies have shown that both are involved in recombination (Tomizawa et al, 1966;Berger et al, 1969;Glassberg et al, 1979;Mosig et al, 1979). Under some conditions these proteins stimulate strand exchange mediated by the RecA (McEntee et al, 1980;Radding, 1982) and UvsX (Formosa and Alberts, 1986;Kodadek, 1990) proteins, but they can also inhibit strand transfer (Kodadek, 1990).…”

Section: Introductionmentioning

confidence: 99%

Single-stranded DNA binding protein and DNA helicase of bacteriophage T7 mediate homologous DNA strand exchange.

Kong

Richardson

1996

The EMBO Journal

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Two proteins encoded by bacteriophage T7, the gene 2.5 single‐stranded DNA binding protein and the gene 4 helicase, mediate homologous DNA strand exchange. Gene 2.5 protein stimulates homologous base pairing of two DNA molecules containing complementary single‐stranded regions. The formation of a joint molecule consisting of circular, single‐stranded M13 DNA, annealed to homologous linear, duplex DNA having 3′‐ or 5′‐single‐stranded termini of approximately 100 nucleotides requires stoichiometric amounts of gene 2.5 protein. In the presence of gene 4 helicase, strand transfer proceeds at a rate of > 120 nucleotides/s in a polar 5′ to 3′ direction with respect to the invading strand, resulting in the production of circular duplex M13 DNA. Strand transfer is coupled to the hydrolysis of a nucleoside 5′‐triphosphate. The reaction is dependent on specific interactions between gene 2.5 protein and gene 4 protein.

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Mutant single-strand binding protein of Escherichia coli: genetic and physiological characterization

Cited by 180 publications

References 28 publications

Topoisomerase III Acts at the Replication Fork To Remove Precatenanes

Topoisomerase III Acts at the Replication Fork To Remove Precatenanes

Devoted to the lagging strand_the chi subunit of DNA polymerase III holoenzyme contacts SSB to promote processive elongation and sliding clamp assembly

Single-stranded DNA binding protein and DNA helicase of bacteriophage T7 mediate homologous DNA strand exchange.

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