Resolution of Holliday intermediates in recombination and DNA repair: indirect suppression of ruvA, ruvB, and ruvC mutations

Mandal, Tikshna N.; Mahdi, Akeel A.; Sharples, Gary J.; Lloyd, Robert G.

doi:10.1128/jb.175.14.4325-4334.1993

Cited by 162 publications

(149 citation statements)

References 47 publications

(58 reference statements)

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“…Genetic analyses indicate that RuvC resolvase plays a defined role in processing recombination and repair intermediates (7)(8)(9)(10)(11). In vitro studies confirm that RuvC specifically interacts with and resolves Holliday junctions by endonucleolytic cleavage (12,13).…”

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confidence: 78%

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

Yoshikawa

Iwasaki

Shinagawa

2001

Journal of Biological Chemistry

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Escherichia coli RuvC resolvase is a specific endonuclease that recognizes and cleaves Holliday junctions formed during homologous recombination and recombinational repair. This study examines the phenotype of RuvC mutants with amino acid substitutions at phenylalanine 69 (F69L, F69Y, F69W, and F69A), a catalytically important residue that faces the catalytic center of the enzyme. F69Y, but not the other three mutants, almost fully complements the UV sensitivity of a ⌬ruvC strain and substantially resolves synthetic Holliday junctions in vitro. In the presence of 100 mM NaCl, RuvC F69A and F69L are defective in junction binding, but F69Y and F69W retain near wild-type binding activity during a gel shift binding assay. KMnO 4 was used to probe synthetic Holliday junction DNA in a complex with wild-type and mutant RuvC; F69A and F69L did not induce disruption of base pairing at the crossover to the same extent as wild-type RuvC. Thus, the aromatic ring of Phe-69 is involved in DNA binding, probably via a stacking interaction with a nucleotide base, and this interaction may induce a structural change in junction DNA that is required to form a catalytically competent complex.

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confidence: 78%

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

Yoshikawa

Iwasaki

Shinagawa

2001

Journal of Biological Chemistry

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“…The following alleles were transduced into FC36 by selection for the indicated drug resistances and screening for increased sensitivity to UV light: recA938:: Tn9200 (45), ⌬(ruvAC) 65, eda57::Cam (18,35), and recG258::dTn10Kan (30). The Lac Ϫ Tet s episome was then mated into these strains by selection for proline prototrophy (Pro ϩ ).…”

Section: Methodsmentioning

confidence: 99%

“…Both Tet s alleles were recessive to Tet r , indicating that they contain mutations in the structural gene for tetracycline resistance, tetA. The two tetA alleles were amplified (the 5Ј primer had the sequence of bp 1574 to 1596, and the 3Ј primer was complementary to bp 2820 to 2838 of the TRN10TETR locus [GenBank accession number J01830]) and sequenced with an ABI 373A sequencer.The following alleles were transduced into FC36 by selection for the indicated drug resistances and screening for increased sensitivity to UV light: recA938:: Tn9200 (45), ⌬(ruvAC) 65, eda57::Cam (18,35), and recG258::dTn10Kan (30). The Lac Ϫ Tet s episome was then mated into these strains by selection for proline prototrophy (Pro ϩ ).…”

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confidence: 99%

Nonadaptive mutations occur on the F' episome during adaptive mutation conditions in Escherichia coli

1997

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One of the most studied examples of adaptive mutation is a strain of Escherichia coli, FC40, that cannot utilize lactose (Lac ؊ ) but that readily reverts to lactose utilization (Lac ؉ ) when lactose is its sole carbon source. Adaptive reversion to Lac ؉ occurs at a high rate when the Lac ؊ allele is on an F episome and conjugal functions are expressed. It was previously shown that nonselected mutations on the chromosome did not appear in the Lac ؊ population while episomal Lac ؉ mutations accumulated, but it remained possible that nonselected mutations might occur on the episome. To investigate this possibility, a second mutational target was created on the Lac ؊ episome by mutation of a Tn10 element, which encodes tetracycline resistance (Tet r ), to tetracycline sensitivity (Tet s ). Reversion rates to Tet r during normal growth and during lactose selection were measured. The results show that nonselected Tet r mutations do accumulate in Lac ؊ cells when those cells are under selection to become Lac ؉ . Thus, reversion to Lac ؉ in FC40 does not appear to be adaptive in the narrow sense of the word. In addition, the results suggest that during lactose selection, both Lac ؉ and Tet r mutations are created or preserved by the same recombination-dependent mechanism.Mutations occurring in nondividing cells have been called adaptive when they allow cells to escape from nonlethal selective pressure (5,7,20). A narrower definition of adaptive mutation is that only useful, not deleterious or neutral, mutations occur during selection (7,12,29). A number of possible examples of such specifically adaptive mutations have been reported (7,13,21,22,24,44). Most current theories for adaptive mutation propose that cells under selection produce genetic variants at random, but these variants are transient, or the cells producing them die, unless a useful mutation occurs and the cells start to grow (3,7,22,43). All of these models predict that nonselected mutations may appear in the successful cells (because all variants that exist when a cell begins to grow will be captured), but nonselected mutations should not appear in the unsuccessful cells (reviewed in reference 12).One of the most studied examples of adaptive mutation is a strain of Escherichia coli, FC40, that cannot utilize lactose (Lac Ϫ ) but that readily reverts to lactose utilization (Lac ϩ ) when lactose is its sole carbon source (5). This high level of adaptive reversion occurs when the Lac Ϫ allele is on an FЈ episome and conjugal functions are expressed (17,19,39). Although mutations in a chromosomal gene, rpoB, do not appear in the Lac Ϫ population while episomal Lac ϩ mutations accumulate (13), the question remained whether nonselected mutations might occur on the episome. Here, I report that, indeed, nonselected mutations at another site on FЈ accumulate in Lac Ϫ cells when those cells are under selection to become Lac ϩ . Thus, any given gene on the episome may be subjected to a highly mutagenic process in cells under nonlethal selective pressure. Although t...

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“…After branch migration, a dimer of RuvC resolves the HJ by making two sequence-specific symmetrical cuts, producing either patched or spliced linear products (4 -6). Genetic studies showed that RuvC cannot function in vivo in the absence of RuvAB (7,8), and it has been proposed that an RuvABC complex, known as the resolvasome, allows RuvC to scan for cleavable sequences (3, 9 -11). RuvA binds to HJs in vitro as one tetramer (complex I) or two tetramers that sandwich the junction (complex II) in a concentration-dependent manner; however, it is not clear whether the RuvAB complex contains one or two tetramers of RuvA in vivo (12)(13)(14)(15)(16)(17)(18)(19)(20)(21).…”

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confidence: 99%

Formation of a Stable RuvA Protein Double Tetramer Is Required for Efficient Branch Migration in Vitro and for Replication Fork Reversal in Vivo

Bradley

Baharoglu

Niewiarowski

et al. 2011

Journal of Biological Chemistry

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In bacteria, RuvABC is required for the resolution of Holliday junctions (HJ) made during homologous recombination. The RuvAB complex catalyzes HJ branch migration and replication fork reversal (RFR). During RFR, a stalled fork is reversed to form a HJ adjacent to a DNA double strand end, a reaction that requires RuvAB in certain Escherichia coli replication mutants. The exact structure of active RuvAB complexes remains elusive as it is still unknown whether one or two tetramers of RuvA support RuvB during branch migration and during RFR. We designed an E. coli RuvA mutant, RuvA2 KaP , specifically impaired for RuvA tetramer-tetramer interactions. As expected, the mutant protein is impaired for complex II (two tetramers) formation on HJs, although the binding efficiency of complex I (a single tetramer) is as wild type. We show that although RuvA complex II formation is required for efficient HJ branch migration in vitro, RuvA2 KaP is fully active for homologous recombination in vivo. RuvA2 KaP is also deficient at forming complex II on synthetic replication forks, and the binding affinity of RuvA2 KaP for forks is decreased compared with wild type. Accordingly, RuvA2 KaP is inefficient at processing forks in vitro and in vivo. These data indicate that RuvA2 KaP is a separation-of-function mutant, capable of homologous recombination but impaired for RFR. RuvA2 KaP is defective for stimulation of RuvB activity and stability of HJ⅐RuvA⅐RuvB tripartite complexes. This work demonstrates that the need for RuvA tetramer-tetramer interactions for full RuvAB activity in vitro causes specifically an RFR defect in vivo.The RuvAB complex is a highly sophisticated molecular machine, which carries out branch migration of Holliday junctions during homologous recombination. RuvA binds specifically to four-armed Holliday junctions (HJ) 4 and guides the assembly of two RuvB hexameric rings onto diametrically opposite arms of the HJ. RuvB, an AAA ϩ ATPase (1), is the motor that drives branch migration of the crossover point (1-3). After branch migration, a dimer of RuvC resolves the HJ by making two sequence-specific symmetrical cuts, producing either patched or spliced linear products (4 -6). Genetic studies showed that RuvC cannot function in vivo in the absence of RuvAB (7, 8), and it has been proposed that an RuvABC complex, known as the resolvasome, allows RuvC to scan for cleavable sequences (3, 9 -11). RuvA binds to HJs in vitro as one tetramer (complex I) or two tetramers that sandwich the junction (complex II) in a concentration-dependent manner; however, it is not clear whether the RuvAB complex contains one or two tetramers of RuvA in vivo (12-21). A RuvAB branch migration complex made of two RuvA tetramers would prevent access of RuvC to the Holliday junction. Whether to form the resolvasome the RuvC dimer displaces one of the two RuvA tetramers present in the RuvAB complex, or whether the RuvC dimer simply binds opposite a single RuvA tetramer present in the complex is a currently unanswered question.In addition to ...

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Resolution of Holliday intermediates in recombination and DNA repair: indirect suppression of ruvA, ruvB, and ruvC mutations

Cited by 162 publications

References 47 publications

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

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

Nonadaptive mutations occur on the F' episome during adaptive mutation conditions in Escherichia coli

Formation of a Stable RuvA Protein Double Tetramer Is Required for Efficient Branch Migration in Vitro and for Replication Fork Reversal in Vivo

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