Alteration by site-directed mutagenesis of the conserved lysine residue in the consensus ATP-binding sequence of the RecB protein of<i>Escherichia coli</i>

Hsieh, Susie; Julin, Douglas A.

doi:10.1093/nar/20.21.5647

Cited by 37 publications

(46 citation statements)

References 32 publications

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“…DNA substrates with short 3Ј-overhangs or blunt duplex ends are barely unwound at all (Fig. 3, green traces), in agreement with the published observations (29,33). However, the presence of just two additional ssDNA nucleotides on the 5Ј-strand (NdeI-cut substrate) is sufficient to reveal a rapid and processive helicase activity that is further enhanced if there are four ssDNA nucleotides on the 5Ј-terminated strand (EcoRI-cut substrate).…”

Section: K177qsupporting

confidence: 89%

“…2C) CD enzyme had suggested that the enzyme did not possess ATPase activity on duplex DNA substrates and, consequently, that it was probably not a DNA helicase (29,33). However, those studies only employed plasmid DNA substrates that had been linearized with restriction enzymes that generate blunt ends or short 3Ј-overhangs.…”

Section: K177qmentioning

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: K177qsupporting

confidence: 89%

Section: K177qmentioning

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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“…These protein caps are thought to block RecBCD binding and subsequent exonucleolytic degradation (Oliver and Goldberg 1977), so that T4 2 + but not T4 2 2 phage form plaques on RecBCD nucleaseproficient strains. Wild type and all RecC tunnel mutants reported here were RecBCD nuclease-proficient by this assay; indicating that RecBCD enzyme was functionally intact, at least for nuclease activity, which requires the helicase and ATPase activities (Hsieh and Julin 1992;Yu et al 1998). Chi activity and recombination proficiency were measured using l red gam mutant phage.…”

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confidence: 78%

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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“…However, the effect of these Lys 464 mutations on opening rate is relatively small (2-4-fold), compared with the large (1-2 orders of magnitude) effect of the analogous mutation in NBF2 (Lys 1250 ) on open burst duration. Furthermore, the large effect of mutations in the corresponding invariant "P-loop" lysines on the hydrolysis rate of other GTPases and ATPases (35)(36)(37) indicates that the hydrolysis rate of Lys 464 mutants is likely to be very low. This discrepancy between the predicted hydrolysis rate and the effect on channel gating led us to propose that ATP hydrolysis at NBF1, while essential for CFTR activity, is not directly coupled to the initiation of each open burst (15).…”

Section: Discussionmentioning

confidence: 99%

Redox Reagents and Divalent Cations Alter the Kinetics of Cystic Fibrosis Transmembrane Conductance Regulator Channel Gating

Harrington¹,

Gunderson²,

Kopito³

1999

Journal of Biological Chemistry

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Gating of the cystic fibrosis Cl؊ channel requires hydrolysis of ATP by its nucleotide binding folds, but how this process controls the kinetics of channel gating is poorly understood. In the present work we show that the kinetics of channel gating and presumably the rate of ATP hydrolysis depends on the species of divalent cation present and the oxidation state of the protein.With Ca 2؉ as the dominant divalent cation instead of Mg 2؉ , the open burst duration of the channel is increased approximately 20-fold, and this change is reversible upon washout of Ca 2؉ . In contrast, "soft" divalent cations such as Cd 2؉ interact covalently with cystic fibrosis transmembrane conductance regulator (CFTR). These metals decrease both opening and closing rates of the channel, and the effects are not reversed by washout. Oxidation of CFTR channels with a variety of oxidants resulted in a similar slowing of channel gating. In contrast, reducing agents had the opposite effect, increasing both opening and closing rates of the channel. In cell-attached patches, CFTR channels exhibit both oxidized and reduced types of gating, raising the possibility that regulation of the redox state of the channel may be a physiological mode of control of CFTR channel activity.The cystic fibrosis transmembrane conductance regulator (CFTR) 1 belongs to the superfamily of ATP-binding cassette transporters, members of which are characterized structurally by two ATP-binding motifs and functionally by their ability to couple ATP hydrolysis to transport of a substrate molecule across a membrane (1, 2). Although a role for CFTR in solute transport has been proposed (3-6), no substrate has yet been identified. CFTR remains unique among ATP-binding cassette transporters in that it couples ATP hydrolysis to the opening of a Cl Ϫ channel (6, 7), reviewed in (8). The role of ATP hydrolysis in the CFTR gating process was inferred initially from studies of CFTR gating in electrophysiological experiments using poorly hydrolyzable ATP analogs and transition state phosphate analogs (9 -12). Direct measurement of ATPase activity in purified reconstituted CFTR has confirmed that it functions as an ATPase, and the measured turnover rate of the phosphorylated form (0.5-1 s Ϫ1 ) is consistent with a model in which opening and closing of the CFTR channel gate is coupled directly to ATP hydrolysis (13).The role of ATP hydrolysis by the two nucleotide-binding folds (NBF1 and NBF2) in mediating channel gating has been elucidated by studies examining CFTR channels individually mutated in each of the NBFs (14 -16). These papers presented models in which two ATP hydrolytic events, one occurring at NBF1 and one at NBF2, are involved first in opening and then closing the CFTR channel. In one model a single cycle of ATP hydrolysis occurs at NBF1 to directly open the channel (8,14,16,17). Alternatively, a second model proposes that ATP hydrolysis at NBF1 converts the channel into a gatable state from which nucleotide binding at NBF2 can then open the channel (15). In both mod...

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Alteration by site-directed mutagenesis of the conserved lysine residue in the consensus ATP-binding sequence of the RecB protein ofEscherichia coli

Cited by 37 publications

References 32 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

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

Redox Reagents and Divalent Cations Alter the Kinetics of Cystic Fibrosis Transmembrane Conductance Regulator Channel Gating

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