Evidence that Phenylalanine 69 in Escherichia coliRuvC Resolvase Forms a Stacking Interaction during Binding and Destabilization of a Holliday Junction DNA Substrate

Yoshikawa, Manabu; Iwasaki, Hiroshi; Shinagawa, Hideo

doi:10.1074/jbc.m010138200

Cited by 24 publications

(21 citation statements)

References 33 publications

(61 reference statements)

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“…In addition to finding the ruvC gene in all of the clinical isolates of H. pylori examined, we also showed that it was present in gastric and enterohepatic Helicobacter species, providing evidence for evolutionary conservation of RuvC across this genus. Although H. pylori RuvC contains all the key residues known to be required by E. coli RuvC for binding and resolution (18,51), it lacks the C-terminal 16-amino-acid extension that may be crucial for interaction with RuvB and stabilization of binding of RuvC on the RuvAB junction complex. This suggests that the functional interactions between the components of the resolvasome structure may be significantly different in H. pylori and E. coli.…”

Section: Discussionmentioning

confidence: 99%

Helicobacter pylori Mutants Defective in RuvC Holliday Junction Resolvase Display Reduced Macrophage Survival and Spontaneous Clearance from the Murine Gastric Mucosa

et al. 2003

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Homologous recombination contributes to the extraordinary genetic diversity of Helicobacter pylori and may be critical for surface antigen expression and adaptation to environmental challenges within the stomach. We generated isogenic, nonpolar H. pylori ruvC mutants to investigate the function of RuvC, a Holliday junction endonuclease that resolves recombinant joints into nicked duplex products. Inactivation of ruvC reduced the frequency of homologous recombination of H. pylori between 17-and 45-fold and increased sensitivity to DNAdamaging agents and the antimicrobial agents levofloxacin and metronidazole. The H. pylori ruvC mutants were more susceptible to oxidative stress and exhibited reduced survival within macrophages. Experiments with the H. pylori SS1 mouse model revealed that the 50% infective dose of the ruvC mutant was approximately 100-fold higher than that of the wild-type SS1 strain. Although the ruvC mutant was able to establish colonization with bacterial loads that were initially similar to those of the parental SS1 strain, infection was spontaneously cleared from the murine gastric mucosa over periods that varied from 36 to 67 days. These results demonstrate that, in this infection model, RuvC is essential for continued survival of H. pylori in vivo and raises the possibility that inactivation of ruvC might be of value in an attenuated vaccine strain.

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Section: Discussionmentioning

confidence: 99%

Helicobacter pylori Mutants Defective in RuvC Holliday Junction Resolvase Display Reduced Macrophage Survival and Spontaneous Clearance from the Murine Gastric Mucosa

et al. 2003

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“…The peptide may inhibit RuvC activity by distorting the central base pairs of the junction and either sterically hindering its binding or interfering with catalysis. The Phe-69 residue of RuvC is involved in DNA binding and catalysis, probably by stacking with a nucleotide near the junction center and producing a conformational change that is necessary for cleavage (43). The peptide prevents binding of RuvC by itself to junctions in the absence of cations (K.V.K.…”

Section: Discussionmentioning

confidence: 99%

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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“…It is also known that the GC or AT skew is useful for identifying the replication origin and strand asymmetry in the prokaryote genome (35), although its application to human genomic sequences is suggestive at best (36). We therefore examined the GC content, the GC and AT skews, and the relative abundance of the consensus sequence 5Ј-(A͞ T)TT(G͞C) or 5Ј-(C͞G)AA(T͞A) at sites where Holliday intermediates in Escherichia coli are resolved (37).…”

Section: Primate Amels Mapped By Fishmentioning

confidence: 99%

The amelogenin loci span an ancient pseudoautosomal boundary in diverse mammalian species

Iwase

Satta

Hirai

et al. 2003

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

124

109

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The mammalian amelogenin (AMEL) genes are found on both the X and Y chromosomes (gametologous). Comparison of the genomic AMEL sequences in five primates and three other mammals reveals that the 5 portion of the gametologous AMEL loci began to differentiate in the common ancestor of extant mammals, whereas the 3 portion differentiated independently within species of different mammals. The boundary is marked by a transposon insertion in intron 2 and is shared by all species examined. In addition, 540-kb DNA sequences from the short arm of the human X chromosome are aligned with their Y gametologous sequences. The pattern and extent of sequence differences in the 5 portion of the AMEL loci extend to a proximal region that contains the ZFX locus, and those in the 3 portion extend all the way down to the pseudoautosomal boundary (PAB)1. We concluded that the AMEL locus spans an ancient PAB, and that both the ancient and present PABs were determined by chance events during the evolution of mammals and primates. Sex chromosome differentiation likely took place in a region that contains the male-determining loci by suppressing homologous recombination.chromosomal rearrangement ͉ evolutionary strata ͉ recombination suppression L ahn and Page (1) have proposed that there are four distinct evolutionary strata on the human X chromosome, and that differentiation of the X from the Y chromosome was initiated one stratum at a time. This hypothesis is based on the observation that the average extent of the sequence divergences at synonymous sites between X and Y homologous, or more precisely gametologous, loci is Ϸ10% in stratum 4 in contrast to 30% in stratum 3, 50% in stratum 2, and 100% in stratum 1. Stratum 4 spans Ϸ20 megabases on the short arm region of the X chromosome and is bounded by the amelogenin (AMEL) locus and pseudoautosomal boundary (PAB)1. Among seven loci examined in stratum 4 (1), AMEL has been more extensively studied in animals other than humans (2-7). Notably, primate intron 3 sequences suggest that AMEL on the X chromosome (AMELX) began to differentiate from that on the Y chromosome (AMELY) before the split of Old World and New World monkeys (4). On the other hand, cDNA or amino acid sequences analysis of gametologous AMELs shows greater relatedness within a species than among different mammalian species (5).Recently, Iwase et al. (8) compared human BAC clones that encompass the AMELX and AMELY loci. They found that, although the region downstream from intron 2 exhibits Ϸ10% sequence differences per site, the upstream region exhibits a high level that is similar to stratum 3 (Ͼ 20%; Ϸ30% if multiple-hit substitutions are taken into account). This finding does not contradict previous results (4, 5), because AMEL exons 1 and 2 almost exclusively encode the 5Ј untranslated region and are excluded from comparisons of intron 3 or amino acid sequences. Therefore, Iwase et al. (8) pointed out that the boundary between strata 3 and 4 on the human X chromosome lies in AMEL intron 2. Their preliminary study of ge...

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Evidence that Phenylalanine 69 in Escherichia coliRuvC Resolvase Forms a Stacking Interaction during Binding and Destabilization of a Holliday Junction DNA Substrate

Cited by 24 publications

References 33 publications

Helicobacter pylori Mutants Defective in RuvC Holliday Junction Resolvase Display Reduced Macrophage Survival and Spontaneous Clearance from the Murine Gastric Mucosa

Helicobacter pylori Mutants Defective in RuvC Holliday Junction Resolvase Display Reduced Macrophage Survival and Spontaneous Clearance from the Murine Gastric Mucosa

Holliday junction-binding peptides inhibit distinct junction-processing enzymes

The amelogenin loci span an ancient pseudoautosomal boundary in diverse mammalian species

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