Efficiency of ATP Hydrolysis and DNA Unwinding by the RecBC Enzyme from Escherichia coli

Korangy, Firouzeh; Julin, Douglas A.

doi:10.1021/bi00198a022

Cited by 53 publications

(74 citation statements)

References 35 publications

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“…RecBCD has been shown to hydrolyze ϳ2 ATP molecules per base pair unwound under optimal conditions, whereas the RecBC and RecBCD K177Q enzymes hydrolyze a little more than 1 ATP per base pair (30,57,58). These results suggest the possibility that each SF1 motor subunit hydrolyzes 1 ATP per base translocated, as has been suggested for the PcrA helicase (26,27).…”

Section: K29qmentioning

confidence: 66%

“…Our study has employed mutants of RecBCD that eliminate active ssDNA translocation in each subunit, but which are not expected to eliminate ssDNA binding activity (43). It should be noted that the lysine to glutamine substitution used here appears to prevent ATP binding rather than ATP hydrolysis per se (32), and that this may influence ssDNA binding allosterically. Electron-microscopic analyses of the unwinding intermediates formed by these mutant proteins suggest that the inactive motor remains bound to the non-translocated strand close to the initiation site for unwinding (22).…”

Section: Discussionmentioning

confidence: 99%

“…RecB K29Q CD, RecBCD K177Q , and singlestranded DNA-binding protein (SSB) were expressed and purified as described (31)(32)(33)(34). Purified proteins are shown in supplementary Fig.…”

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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: 66%

Section: Discussionmentioning

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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“…This model is supported by the fact that recD mutants are recombination-proficient (11,23,29) and hyperre-combination-proficient in the absence of Chi sites (11), and they cluster recombination events at the ends of DNA in lambda replication-blocked crosses (30). Purified RecBC enzyme (i.e., lacking RecD) has a low affinity for dsDNA (unpublished data, see Table 5) ends but can unwind DNA (16,31,32) and facilitates loading of RecA protein at the 3Ј termini of DNA during unwinding (16). This is in contrast to RecBCD enzyme, which requires a Chi site for significant loading of RecA protein (6), joint molecule formation (7), and recombination (18).…”

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confidence: 98%

The RecD subunit of the Escherichia coli RecBCD enzyme inhibits RecA loading, homologous recombination, and DNA repair

Amundsen

Taylor

Smith

2000

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

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. We tested the hypothesis that the RecD subunit inhibits recombination by deleting recD from the nuclease-and recombination-deficient mutant recB D1080A CD. We report here that the resulting strain, recB D1080A C, was proficient for recombination and DNA repair. Recombination proficiency was accompanied by a change in enzyme activity: RecB D1080A C enzyme loaded RecA protein onto DNA during DNA unwinding whereas RecB D1080A CD enzyme did not. Together, these genetic and biochemical results demonstrate that RecA loading by RecBCD enzyme is required for recombination in E. coli cells and suggest that RecD interferes with the enzyme domain required for its loading. A nuclease-dependent signal appears to be required for a change in RecD that allows RecA loading. Because RecA loading is not observed with wild-type RecBCD enzyme until it acts at a Chi site, our observations support the view that RecD inhibits recombination until the enzyme acts at Chi. R ecBCD enzyme plays a central role in the major pathway of genetic exchange and DNA repair in Escherichia coli (1-3). The degradative and recombinational activities of RecBCD enzyme, as well as its structure, are regulated by a specific DNA sequence called Chi (5Ј-GCTGGTGG-3Ј). As a consequence of the RecBCD enzyme-Chi interaction, both the DNA substrate (4-7) and enzyme (8-10) are changed. Genetic and biochemical experiments have suggested that one or another RecBCD enzyme subunit directs this regulation (8,(11)(12)(13)(14)(15)(16). We show here that the RecD subunit inhibits E. coli recombination by blocking RecBCD enzyme-facilitated loading of RecA protein onto single-stranded (ss) DNA.The structure of the RecBCD enzyme and the activities it promotes are complex. RecBCD enzyme is a heterotrimer composed of one copy of each of the products of the recB, recC, and recD genes (17). The enzyme is an ATP-dependent doublestranded (ds) and ss exonuclease, a ss endonuclease, and a DNA helicase (1). RecBCD enzyme interacts with Chi sites, which stimulate recombination in E. coli and bacteriophage lambda (18). Null mutations in recB and recC result in recombination deficiency and sensitivity to DNA damaging agents (19)(20)(21). Cultures of such strains contain many inviable cells (22), reflecting their inability to repair DNA damage by homologous recombination. In contrast, null mutations in recD leave strains highly viable and proficient in recombination and DNA repair (refs. 11 and 23; see below).The role of RecBCD enzyme in homologous recombination begins when it binds to the end of a dsDNA substrate and initiates unwinding. Further reactions of RecBCD enzyme with DNA occur in a manner dependent on the presence of Chi sites and the concentrations of Mg 2ϩ and ATP. When the concentration of ATP exceeds that of Mg 2ϩ , RecBCD enzyme makes a ss endonucleolytic cut a few nt to the 3Ј side of Chi (4, 5). When the concentration of Mg 2ϩ exceeds that of ATP, RecBCD enzyme degrades the 3Ј terminated strand during DNA unwinding; the degradation is reduced when the enzyme reaches Ch...

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“…A Walker A box is defined by the consensus sequence (G/A)XXGXGKT (X is any amino acid (29). RecBCD enzymes in which the con-served Lys in this motif is changed to Gln have a reduced affinity for ATP binding (33,34) and altered helicase activity (17,(35)(36)(37).…”

mentioning

confidence: 99%

Dual Nuclease and Helicase Activities of Helicobacter pylori AddAB Are Required for DNA Repair, Recombination, and Mouse Infectivity

Amundsen

Fero

Salama

et al. 2009

Journal of Biological Chemistry

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Helicobacter pylori infection of the human stomach is associated with disease-causing inflammation that elicits DNA damage in both bacterial and host cells. NUC showed partial attenuation. E. coli ⌬recBCD expressing H. pylori addAB was recombination-deficient unless H. pylori recA was also expressed, suggesting a species-specific interaction between AddAB and RecA and also that H. pylori AddAB participates in both DNA repair and recombination. These results support a role for both the AddAB nuclease and helicase in DNA repair and promoting infectivity.

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Efficiency of ATP Hydrolysis and DNA Unwinding by the RecBC Enzyme from Escherichia coli

Cited by 53 publications

References 35 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

The RecD subunit of the Escherichia coli RecBCD enzyme inhibits RecA loading, homologous recombination, and DNA repair

Dual Nuclease and Helicase Activities of Helicobacter pylori AddAB Are Required for DNA Repair, Recombination, and Mouse Infectivity

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