Structural Analysis of DNA Replication Fork Reversal by RecG

Singleton, Martin R.; Scaife, Sarah; Wigley, Dale B.

doi:10.1016/s0092-8674(01)00501-3

Cited by 279 publications

(360 citation statements)

References 56 publications

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“…The HJ center is slightly more susceptible to KMnO 4 cleavage in the presence of RecG alone (Fig. 2 E and F), consistent with crystal structure data indicating that the wedge domain of RecG interacts with the junction point (29). The presence of both RecG and peptide WRWYCR in the binding reaction results in a KMnO 4 cleavage pattern that closely matches the 3-to 4-fold increase in KMnO 4 sensitivity induced by peptide alone (Fig.…”

Section: Resultssupporting

confidence: 84%

“…The crystal structure of RecG bound to a fork substrate suggests how competition may occur (29). The 98-aa wedge domain of RecG makes intimate contact with the replication fork substrate, inserting between the two arms of the replication fork.…”

Section: Resultsmentioning

confidence: 99%

“…The 98-aa wedge domain of RecG makes intimate contact with the replication fork substrate, inserting between the two arms of the replication fork. In the crystal structure, Phe-204 and Tyr-208 of RecG contact the central bases in a manner that mimics base stacking and probably provide stacking partners for bases at the junction point during unwinding (29). The peptides may occupy the same space as the RecG wedge domain, causing distortion of the central bases and disrupting interactions between the HJ or replication fork substrate and RecG.…”

Section: Resultsmentioning

confidence: 99%

“…Aromatic amino acids in the peptide may stack with the central base pairs in the junction, rendering these more susceptible to oxidation by permanganate. Such stacking interactions may resemble those seen between two aromatic amino acids present in the wedge domain of the Thermotoga maritima RecG protein and nucleotides in the center of its replication fork substrate (29). Such interactions would be possible only with junctions in the extended, or open, conformation.…”

Section: Discussionmentioning

confidence: 99%

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Holliday junction-binding peptides inhibit distinct junction-processing enzymes

Kepple

Boldt

Segall

2005

Proc. Natl. Acad. Sci. U.S.A.

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Holliday junctions (HJ) are the central intermediates in both homologous recombination and site-specific recombination performed by tyrosine recombinases such as the bacteriophage Integrase (Int) protein. Previously, our lab identified peptide inhibitors of Int-mediated recombination that prevent the resolution of HJ intermediates. We now show that two of these inhibitors bind HJ DNA in the square-planar conformation even in the absence of Int protein. The peptides prevent unwinding of branched DNA substrates by the RecG helicase of Escherichia coli and interfere with the resolution of HJ substrates by the RuvABC complex. Our results suggest that these peptides target all proteins that process HJ in the square-planar conformation. These inhibitors should be extremely useful for dissecting homologous recombination and recombination-dependent repair in vitro and in vivo.homologous recombination ͉ recombination-dependent repair ͉ RecG helicase ͉ RuvABC resolvasome ͉ tyrosine recombinase H olliday junctions (HJ), or four-way junctions, are central intermediates in homologous recombination, repair of collapsed replication forks, and reactions performed by the tyrosine recombinase family of site-specific enzymes (1-5). The first two processes are important in all organisms and are involved in the maintenance of chromosome integrity and repair of DNA damage. In the case of diploid organisms, faithful chromosome segregation depends on homologous recombination (6). Sitespecific recombination reactions performed by tyrosine recombinases are also very widespread and control gene expression, regulate plasmid and bacterial chromosome copy number, and mediate lysogeny (3). The presence and disappearance of HJ, their level within cells, and the enzymes that both generate and resolve them are of intense interest. More tools would be extremely useful for both in vitro and in vivo dissection of homologous recombination and repair processes in all organisms.Phage integrase (Int) binds to and mediates strand exchange between pairs of att sites. During recombination, one round of DNA cleavage, strand exchange, and ligation of the top strands of each partner DNA molecule generates a HJ intermediate, which is resolved by a second round of the same catalytic steps (3). These reactions are both rapid and highly reversible, making intermediates very difficult to study.We have identified peptides that inhibit recombination by trapping the protein-bound HJ intermediate and preventing its resolution either to substrates or to products (7-9). The most potent inhibitory peptide, WRWYCR, traps virtually all HJ formed during a reaction and has an IC 50 of 5-20 nM (9). A related peptide, KWWCRW, is very similar to WRWYCR in potency (8). These peptides also inhibit the mechanistically related Cre, XerC and D, and Flp proteins (ref. 9; A.M.S., unpublished results; A. Conway and P. A. Rice, personal communication). Because these proteins share little primary sequence identity, we reasoned that these peptides might interact with free HJ.HJ adopt one...

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Section: Resultssupporting

confidence: 84%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Holliday junction-binding peptides inhibit distinct junction-processing enzymes

Kepple

Boldt

Segall

2005

Proc. Natl. Acad. Sci. U.S.A.

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“…The bound ssDNA is in a configuration more similar to that found in a DNA duplex, with the bases stacked against one another, unlike the extended conformation observed in PcrA. Interestingly, the RecD2 mode of binding is more similar to that seen in SF2 enzymes such as NS3, Rad54, Vasa, and Hel308 [104,[123][124][125] rather than SF1A helicases [126].…”

Section: Structure Of Sf1b Helicases (Recd2 and Dda)mentioning

confidence: 85%

Erratum to: Structure and Mechanisms of SF1 DNA Helicases

Raney

Byrd

Aarattuthodiyil

2012

Advances in Experimental Medicine and Biology

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Superfamily I is a large and diverse group of monomeric and dimeric helicases defined by a set of conserved sequence motifs. Members of this class are involved in essential processes in both DNA and RNA metabolism in all organisms. In addition to conserved amino acid sequences, they also share a common structure containing two RecA-like motifs involved in ATP binding and hydrolysis and nucleic acid binding and unwinding. Unwinding is facilitated by a "pin" structure which serves to split the incoming duplex. This activity has been measured using both ensemble and single-molecule conditions. SF1 helicase activity is modulated through interactions with other proteins.

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RecG controls DNA amplification at double‐strand breaks and arrested replication forks

Azeroglu

Leach

2017

FEBS Letters

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DNA amplification is a powerful mutational mechanism that is a hallmark of cancer and drug resistance. It is therefore important to understand the fundamental pathways that cells employ to avoid over‐replicating sections of their genomes. Recent studies demonstrate that, in the absence of RecG, DNA amplification is observed at sites of DNA double‐strand break repair (DSBR) and of DNA replication arrest that are processed to generate double‐strand ends. RecG also plays a role in stabilising joint molecules formed during DSBR. We propose that RecG prevents a previously unrecognised mechanism of DNA amplification that we call reverse‐restart, which generates DNA double‐strand ends from incorrect loading of the replicative helicase at D‐loops formed by recombination, and at arrested replication forks.

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Structural Analysis of DNA Replication Fork Reversal by RecG

Cited by 279 publications

References 56 publications

Holliday junction-binding peptides inhibit distinct junction-processing enzymes

Holliday junction-binding peptides inhibit distinct junction-processing enzymes

Erratum to: Structure and Mechanisms of SF1 DNA Helicases

RecG controls DNA amplification at double‐strand breaks and arrested replication forks

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