The 30-kDa C-terminal domain of the RecB protein is critical for the nuclease activity, but not the helicase activity, of the  RecBCD enzyme from 
            <i>Escherichia coli</i>

Yu, Misook; Souaya, Jehanne; Julin, Douglas A.

doi:10.1073/pnas.95.3.981

Cited by 109 publications

(118 citation statements)

References 42 publications

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“…Therefore, these studies clearly indicate that the essential functional unit required for DNA helicase activity catalyzed by a SF1 enzyme can be encoded by a single set of SF1 helicase motifs. -It is also of interest that the observed unwinding rates and processivities of the RecB and RecD helicases are very much poorer when in isolation, compared with when they are found in the context of the holoenzyme (21,24,49). 4 This suggests that the architecture of the complete heterotrimeric molecular machine is vital to harness the ssDNA motor activity of each subunit and translate it into effective DNA unwinding.…”

Section: K29qmentioning

confidence: 99%

“…The RecC subunit also plays a role, because the RecBC enzyme is a fast and processive helicase, whereas RecB is poorly processive (49). The crystal structure of RecBCD suggests an obvious structural basis for this stimulatory effect of RecC; the RecC subunit forms tunnels for each of the unwound ssDNA strands as they pass through the RecBCD complex (45).…”

Section: K29qmentioning

confidence: 99%

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Bipolar DNA Translocation Contributes to Highly Processive DNA Unwinding by RecBCD Enzyme

Dillingham

Webb²,

Kowalczykowski

2005

Journal of Biological Chemistry

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We recently demonstrated that the RecBCD enzyme is a bipolar DNA helicase that employs two single-stranded DNA motors of opposite polarity to drive translocation and unwinding of duplex DNA. We hypothesized that this organization may explain the exceptionally high rate and processivity of DNA unwinding catalyzed by the RecBCD enzyme. Using a stopped-flow dye displacement assay for unwinding activity, we test this idea by analyzing mutant RecBCD enzymes in which either of the two helicase motors is inactivated by mutagenesis. Like the wild-type RecBCD enzyme, the two mutant proteins maintain the ability to bind tightly to blunt duplex DNA ends in the absence of ATP. However, the rate of forward translocation for the RecB motor-defective enzyme is only ϳ30% of the wild-type rate, whereas for the RecD motor-defective enzyme, it is ϳ50%. More significantly, the processivity of translocation is substantially reduced by ϳ25-and 6-fold for each mutant enzyme, respectively. Despite retaining the capacity to bind blunt dsDNA, the RecB-mutant enzyme has lost the ability to unwind DNA unless the substrate contains a short 5-terminated singlestranded DNA overhang. The consequences of this observation for the architecture of the single-stranded DNA motors in the initiation complex are discussed.The RecBCD enzyme of Escherichia coli is involved in the repair of double-stranded breaks in DNA (for review, see Ref. 1). This is an important function, because it has recently become apparent that double-stranded breaks occur frequently as a result, among other things, of replication through damaged template DNA (for discussions, see Refs. 2 and 3). The break in the nucleic acid is rescued by homologous recombination, which uses an undamaged copy of the DNA molecule as a template for the repair. Recombinational DNA repair involves several gene products, and the RecBCD helicase/nuclease acts at the initiation step. The enzyme binds tightly to blunt or nearly blunt DNA ends (4, 5). Then, in a reaction that requires ATP hydrolysis, RecBCD rapidly tracks along the duplex DNA, unwinding it as it goes, and predominantly degrading the 3Ј-terminated single strand into shorter fragments. This destructive, nucleolytic mode of action is modified when the enzyme encounters a correctly oriented DNA sequence called Chi (crossover hotspot instigator 5Ј-GCTGGTGG). This important regulatory sequence is over-represented in the E. coli genome and is particularly found clustered, with appropriate directionality, around the origin of replication (6, 7). Upon Chi recognition, degradation of the 3Ј-terminated strand is reduced, and degradation of the 5Ј-strand is up-regulated, although less so, while DNA translocation and unwinding continue (8 -10). Consequently, the product of the enzyme is a DNA duplex with a long 3Ј-terminated ssDNA 3 overhang onto which the enzyme loads RecA protein (11) to create a recombinogenic molecule, ready for the subsequent DNA strand exchange step of homologous recombination (3). In the absence of Chi recognition, RecBCD cont...

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Section: K29qmentioning

confidence: 99%

Section: K29qmentioning

confidence: 99%

Bipolar DNA Translocation Contributes to Highly Processive DNA Unwinding by RecBCD Enzyme

Dillingham

Webb²,

Kowalczykowski

2005

Journal of Biological Chemistry

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“…indicates that the RecD subunit plays an essential role in DNA degradation. However, analysis of mutations in the RecB subunit establishes that the C-terminal domain of this enzyme is absolutely essential for all nuclease activities of the RecBCD enzyme (25,26). Furthermore, fusion of the C-terminal domain of the RecB subunit with the DNA binding domain of T4 phage gene 32 protein creates a nonspecific nuclease, showing that the C terminus of RecB subunit is indeed a nuclease.…”

mentioning

confidence: 99%

A Single Mutation, RecBD1080A, Eliminates RecA Protein Loading but Not Chi Recognition by RecBCD Enzyme

Anderson

Churchill

Kowalczykowski

1999

Journal of Biological Chemistry

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Homologous recombination and double-stranded DNA break repair in Escherichia coli are initiated by the multifunctional RecBCD enzyme. After binding to a double-stranded DNA end, the RecBCD enzyme unwinds and degrades the DNA processively. This processing is regulated by the recombination hot spot, Chi (: 5-GCT-GGTGG-3), which induces a switch in the polarity of DNA degradation and activates RecBCD enzyme to coordinate the loading of the DNA strand exchange protein, RecA, onto the single-stranded DNA products of unwinding. Recently, a single mutation in RecB, Asp-1080 3 Ala, was shown to create an enzyme (RecB D1080A CD) that is a processive helicase but not a nuclease. Here we show that the RecB D1080A CD enzyme is also unable to coordinate the loading of the RecA protein, regardless of whether sites are present in the DNA. However, the RecB D1080A CD enzyme does respond to sites by inactivating in a -dependent manner. These data define a locus of the RecBCD enzyme that is essential not only for nuclease function but also for the coordination of RecA protein loading.

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“…recD null mutants are Rec 1 , are barely more sensitive to DNA-damaging agents than wild type, and form large colonies. In these mutants, Chi is inactive (Chi À ), and the resultant enzyme, called RecBC, is insensitive to Chi (Chaudhury and Smith 1984b;Amundsen et al 1986;Yu et al 1998a). This phenotype, Chi…”

Section: Resultsmentioning

confidence: 99%

“…Nevertheless, RecBC enzyme has ,1/1000 of the ds and ss exonuclease activity of the wild-type enzyme (Yu et al 1998a). Thus, RecD must regulate the nuclease activity.…”

Section: Discussionmentioning

confidence: 99%

Chi Hotspot Activity in Escherichia coli Without RecBCD Exonuclease Activity: Implications for the Mechanism of Recombination

Amundsen

Smith

2007

Genetics

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The major pathway of genetic recombination and DNA break repair in Escherichia coli requires RecBCD enzyme, a complex nuclease and DNA helicase regulated by Chi sites (59-GCTGGTGG-39). During its unwinding of DNA containing Chi, purified RecBCD enzyme has two alternative nucleolytic reactions, depending on the reaction conditions: simple nicking of the Chi-containing strand at Chi or switching of nucleolytic degradation from the Chi-containing strand to its complement at Chi. We describe a set of recC mutants with a novel intracellular phenotype: retention of Chi hotspot activity in genetic crosses but loss of detectable nucleolytic degradation as judged by the growth of mutant T4 and l phages and by assay of cell-free extracts. We conclude that RecBCD enzyme's nucleolytic degradation of DNA is not necessary for intracellular Chi hotspot activity and that nicking of DNA by RecBCD enzyme at Chi is sufficient. We discuss the bearing of these results on current models of RecBCD pathway recombination.

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The 30-kDa C-terminal domain of the RecB protein is critical for the nuclease activity, but not the helicase activity, of the RecBCD enzyme from Escherichia coli

Cited by 109 publications

References 42 publications

Bipolar DNA Translocation Contributes to Highly Processive DNA Unwinding by RecBCD Enzyme

Bipolar DNA Translocation Contributes to Highly Processive DNA Unwinding by RecBCD Enzyme

A Single Mutation, RecBD1080A, Eliminates RecA Protein Loading but Not Chi Recognition by RecBCD Enzyme

Chi Hotspot Activity in Escherichia coli Without RecBCD Exonuclease Activity: Implications for the Mechanism of Recombination

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