χ*, a χ‐related 11‐mer sequence partially active in an <i>E. coli recC</i>* strain

Handa, Nobuhiko; Ohashi, Seishi; Kusano, Kohji; Kobayashi, Ichizo

doi:10.1046/j.1365-2443.1997.1410339.x

Cited by 45 publications

(69 citation statements)

References 34 publications

(52 reference statements)

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“…The first reported mutants that blocked Chi activation, but retained RecBCD enzymatic activities and recombination proficiency, were pseudorevertants of the recC73 null allele, as noted in the Introduction . One of these mutants, recC1004, has no detectable activity with standard Chi sites in l (x + 76, x + C157, and x + D123) ) but does have partial activity with the x* sequence 59 GCTGGTGCTCG 39 noted above (Handa et al 1997;Arnold et al 2000). This sequence confers large-plaque formation and increased burst size of l red gam phage in both recC1004 mutant and recBCD + cells, but it does not produce recombination hotspot activity detectable in either strain.…”

Section: Molecular Basis Of Recbcd's Recognition Of Chi and Its Contementioning

confidence: 98%

“…It remained possible, however, that Chi in a given context is active with purified proteins but not as a recombination hotspot in cells. For example, a Chi-related sequence (59 GCTGGTGCTCG 39), denoted x*, weakly stimulates cutting of DNA and loading of RecA protein by the purified RecBC 1004 D pseudorevertant mutant and wild-type RecBCD enzymes, but x* has no detectable recombination hotspot activity in cells, either recBCD + or recC1004, a frameshift pseudorevertant with eight amino acid changes in the RecC tunnel Handa et al 1997;Arnold et al 2000). With this exception, the published data were consistent with both the genetic and biochemical activities of Chi being determined simply by 59 GCTGGTGG 39, but our observations reported here force a revision of this view.…”

Section: Analysis Of Recbcd Mutants Reveals An Additional Feature Of mentioning

confidence: 99%

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RecBCD Enzyme “Chi Recognition” Mutants Recognize Chi Recombination Hotspots in the Right DNA Context

2016

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RecBCD enzyme is a complex, three-subunit protein machine essential for the major pathway of DNA double-strand break repair and homologous recombination in Escherichia coli. Upon encountering a Chi recombination-hotspot during DNA unwinding, RecBCD nicks DNA to produce a single-stranded DNA end onto which it loads RecA protein. Conformational changes that regulate RecBCD's helicase and nuclease activities are induced upon its interaction with Chi, defined historically as 59 GCTGGTGG 39. Chi is thought to be recognized as single-stranded DNA passing through a tunnel in RecC. To define the Chi recognition-domain in RecC and thus the mechanism of the RecBCD-Chi interaction, we altered by random mutagenesis eight RecC amino acids lining the tunnel. We screened for loss of Chi activity with Chi at one site in bacteriophage l. The 25 recC mutants analyzed thoroughly had undetectable or strongly reduced Chi-hotspot activity with previously reported Chi sites. Remarkably, most of these mutants had readily detectable, and some nearly wild-type, activity with Chi at newly generated Chi sites. Like wild-type RecBCD, these mutants had Chi activity that responded dramatically (up to fivefold, equivalent to Chi's hotspot activity) to nucleotide changes flanking 59 GCTGGTGG 39. Thus, these and previously published RecC mutants thought to be Chi-recognition mutants are actually Chi context-dependence mutants. Our results fundamentally alter the view that Chi is a simple 8-bp sequence recognized by the RecC tunnel. We propose that Chi hotspots have dual nucleotide sequence interactions, with both the RecC tunnel and the RecB nuclease domain.KEYWORDS chromosomal sites; recombination hotspots; Chi; RecBCD enzyme; DNA context-dependence H OMOLOGOUS recombination, like other aspects of chromosome metabolism, is controlled by special DNA sites. The sites controlling homologous recombination that are best understood at the molecular level are the Chi hotspots of Escherichia coli (Smith 2012). Chi sites control RecBCD enzyme, a complex enzyme with both nuclease and helicase activities essential for the major pathway of DNA double-strand break repair and genetic recombination. These sites, called Chi for crossover hotspot instigator (Lam et al. 1974), locally stimulate recombination by altering the multiple activities of RecBCD. Here, we address the question of how RecBCD recognizes Chi, the first step in its stimulation of recombination. Our results force a reanalysis of both the Chi nucleotide sequence and how RecBCD recognizes Chi.Chi was discovered as a set of mutations that increase the plaque size of phage l red gam mutants, which rely on the E. coli RecBCD pathway for growth (Lam et al. 1974;McMilin et al. 1974;Henderson and Weil 1975). In the absence of its own recombination functions (Red) and of the RecBCD inhibitor (Gam), l replication forms only monomeric circles. Phage DNA packaging, however, requires dimeric or higher order concatemers, which can be formed by RecBCDpromoted recombination between monomers. Wild-typ...

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Section: Molecular Basis Of Recbcd's Recognition Of Chi and Its Contementioning

confidence: 98%

Section: Analysis Of Recbcd Mutants Reveals An Additional Feature Of mentioning

confidence: 99%

RecBCD Enzyme “Chi Recognition” Mutants Recognize Chi Recombination Hotspots in the Right DNA Context

2016

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“…Limited proteolysis of the RecC protein suggests that a 35-kDa C-terminal domain is required for interaction with the RecD protein (9). Certain mutations in the RecC protein are responsible for RecBCD holoenzymes with altered Chi recognition specificity, arguing that the RecC protein is involved in Chi recognition (22,124); we will return to this point in the discussion of the RecBCD-DNA crystal structure.…”

Section: Isolated Subunitsmentioning

confidence: 99%

“…Thirdly, the 3Ј tunnel in RecC is, in structural terms, a nonfunctional equivalent of a helicase motor (it is formed by the core helicase domains 1A and 2A), which might provide an ideal protein architecture to function as a scanning site for a specific ssDNA sequence. Finally, members of the RecC* class of mutations, which alter the efficiency and specificity of Chi recognition, map to the 3Ј tunnel in the RecC subunit (22,124,248) The RecC protein also appears to assist in the DNA-unwinding activity by contributing a dual-methionine "pin" that acts as a wedge to split the duplex at the junction between ssDNA and dsDNA (Fig. 13).…”

Section: Vol 72 2008mentioning

confidence: 99%

RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks

Dillingham

Kowalczykowski

2008

Microbiol Mol Biol Rev

503

599

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SUMMARY The RecBCD enzyme of Escherichia coli is a helicase-nuclease that initiates the repair of double-stranded DNA breaks by homologous recombination. It also degrades linear double-stranded DNA, protecting the bacteria from phages and extraneous chromosomal DNA. The RecBCD enzyme is, however, regulated by a cis-acting DNA sequence known as Chi (crossover hotspot instigator) that activates its recombination-promoting functions. Interaction with Chi causes an attenuation of the RecBCD enzyme's vigorous nuclease activity, switches the polarity of the attenuated nuclease activity to the 5′ strand, changes the operation of its motor subunits, and instructs the enzyme to begin loading the RecA protein onto the resultant Chi-containing single-stranded DNA. This enzyme is a prototypical example of a molecular machine: the protein architecture incorporates several autonomous functional domains that interact with each other to produce a complex, sequence-regulated, DNA-processing machine. In this review, we discuss the biochemical mechanism of the RecBCD enzyme with particular emphasis on new developments relating to the enzyme's structure and DNA translocation mechanism.

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“…In lambdoid bacteriophages, packaging of the phage genome into a viable phage particle needs a concatemer form, in which phage DNA units are joined together in a head-to-tail manner (Fujisawa and Morita 1997). Formation of the concatemer is blocked by the RecBCD DNase of the host E. coli, which degrades nonself DNA but repairs self DNA marked by an ID sequence (Handa et al 1997(Handa et al , 2000. Lambda and other bacteriophages produce an inhibitor of RecBCD DNase (Smith 1983).…”

Section: Discussionmentioning

confidence: 99%

Evolution of DNA Double-Strand Break Repair by Gene Conversion: Coevolution Between a Phage and a Restriction-Modification System

et al. 2007

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The necessity to repair genome damage has been considered to be an immediate factor responsible for the origin of sex. Indeed, attack by a cellular restriction enzyme of invading DNA from several bacteriophages initiates recombinational repair by gene conversion if there is homologous DNA. In this work, we modeled the interaction between a bacteriophage and a bacterium carrying a restriction enzyme as antagonistic coevolution. We assume a locus on the bacteriophage genome has either a restriction-sensitive or a restriction-resistant allele, and another locus determines whether it is recombination/repair proficient or defective. A restriction break can be repaired by a co-infecting phage genome if one of them is recombination/ repair proficient. We define the fitness of phage (resistant/sensitive and repair-positive/-negative) genotypes and bacterial (restriction-positive/-negative) genotypes by assuming random encounter of the genotypes, with given probabilities of single and double infections, and the costs of resistance, repair, and restriction. Our results show the evolution of the repair allele depends on b 1 =b 0 , the ratio of the burst size b 1 under damage to host cell physiology induced by an unrepaired double-strand break to the default burst size b 0 . It was not until this effect was taken into account that the evolutionary advantage of DNA repair became apparent.

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χ, a χ‐related 11‐mer sequence partially active in an E. coli recC** strain

Cited by 45 publications

References 34 publications

RecBCD Enzyme “Chi Recognition” Mutants Recognize Chi Recombination Hotspots in the Right DNA Context

RecBCD Enzyme “Chi Recognition” Mutants Recognize Chi Recombination Hotspots in the Right DNA Context

RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks

Evolution of DNA Double-Strand Break Repair by Gene Conversion: Coevolution Between a Phage and a Restriction-Modification System

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χ*, a χ‐related 11‐mer sequence partially active in an E. coli recC* strain

Cited by 45 publications

References 34 publications

RecBCD Enzyme “Chi Recognition” Mutants Recognize Chi Recombination Hotspots in the Right DNA Context

RecBCD Enzyme “Chi Recognition” Mutants Recognize Chi Recombination Hotspots in the Right DNA Context

RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks

Evolution of DNA Double-Strand Break Repair by Gene Conversion: Coevolution Between a Phage and a Restriction-Modification System

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χ, a χ‐related 11‐mer sequence partially active in an E. coli recC** strain