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

Abstract

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...

Characterization of the Mycobacterial AdnAB DNA Motor Provides Insights into the Evolution of Bacterial Motor-Nuclease Machines

“…The RecB subunit engages and translocates along the 3Ј DNA strand during duplex unwinding while the RecD subunit translocates along the 5Ј DNA strand (24,25). By analogy to an electrical circuit, parallel motors hydrolyze ATP independently, so that mutation of the RecD phosphohydrolase active site does not preclude ATP hydrolysis and DNA translocation by RecB; in turn, mutation of the RecB phosphohydrolase site does not prevent ATP hydrolysis and DNA translocation by RecD (33,34). By contrast, mycobacterial AdnAB seems to behave like a serial motor, in which ATP hydrolysis by the leading (dominant) AdnB subunit precedes ATP hydrolysis by the lagging (dependent) AdnA subunit.…”

RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks

“…In the first scenario, the leading motor is acting as a true DNA helicase, while the second slower motor is simply an ssDNA translocase. However, it is important to note that this distinction is superficial because experiments with mutants containing only one active motor subunit clearly demonstrate that either RecB or RecD can act alone as an efficient DNA helicase in the context of the holoenzyme (88). This experiment also demonstrates that the two motors are (at least substantially) autonomous.…”

“…ATP hydrolysis is strongly dependent on linear ssDNA or dsDNA, is extremely fast (ϳ1,000 bp s Ϫ1 at 25°C) (237), and supports a rapid and processive DNA-unwinding activity (238). To our knowledge, the RecBCD enzyme remains the fastest (1,000 to 2,000 bp s Ϫ1 ) (31,88,97,120,237,238,266,267) and most processive (ϳ30,000 bp) (31,88,120,236) bona fide helicase reported in the literature. Importantly, DNA unwinding by the RecBCD holoenzyme is substantially faster and more processive than DNA unwinding by either the purified RecB or RecD subunit alone (87) or the isolated helicase subunits of closely related proteins such as PcrA, Rep, and UvrD (see references 88 and 257 for discussion).…”

“…First, RecD is needed to "activate" the nucleolytic functions contained within the RecB subunit, since the RecBC enzyme lacking the RecD subunit has comparably little nuclease activity (12,152,154). Second, RecD contributes to the processivity of the holoenzyme since its processivity is higher than that of the RecBC enzyme (88,154). Third, as mentioned above, it also negatively regulates the RecA-loading ability of the RecBCD enzyme (10).…”

Kinetics of DNA Unwinding by the RecD2 Helicase from Deinococcus radiodurans

“…Despite its low DNA unwinding activity in isolation, the RecD subunit contributes to rapid DNA unwinding by RecBCD. A RecBCD mutant enzyme in which the RecB subunit is inactive, and which therefore depends on RecD for translocation on the DNA, still unwinds DNA very rapidly (480 bp/s), about one-third as fast as the wild-type enzyme (38). Thus the unwinding activity of the RecD subunit is greatly enhanced by assembly with RecB and RecC to make RecBCD.…”