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  • RecBCD is a combination helicase nuclease


    RecBCD is a combination helicase/nuclease that is critical to homologous genetic recombination and DNA repair in E. coli., The holoenzyme consists of the three subunits RecB, RecC, and RecD. The RecB and RecD subunits are NTP hydrolysis-dependent DNA motor proteins that drive DNA translocation and strand separation,, while RecC functions in holoenzyme scaffolding, DNA unwinding and recognition of the recombination hotspot χ. Together, these subunits form a complex enzyme possessing DNA helicase, ATPase and nuclease activities. In vitro, RecBCD binds to blunt or almost blunt dsDNA, ends with >106-fold affinity relative to internal sites. The helicase unwinds the DNA at rates of up to ∼1000 bp s−1 at 37 °C, traversing on average 30,000 bp per binding event., DNA unwinding is accompanied by the simultaneous and asymmetric endonucleolytic degradation of unwound ssDNA with the 3′-terminated strand, relative to the entry point of the enzyme, being preferentially degraded (Figure 1(a)). For both translocation and DNA degradation to occur, RecBCD requires Mg2+ and the R406 derived from the hydrolysis of a nucleoside triphosphate, typically ATP. The nuclease activity of RecBCD is regulated by the recombination hotspot χ (chi for crossover hotspot instigator; defined as 5′-GCTGGTGG-3′). The translocating RecBCD enzyme recognizes χ as the unwound single strand of DNA but only when approaching χ from the 3′-side (Figure 1(b)) Recognition of χ results in the translocating enzyme pausing at χ, during which the polarity of the nuclease activity is altered. The 3′ to 5′ activity is attenuated while the 5′ to 3′ nuclease activity is up-regulated. Consequently, continued translocation past χ results in RecBCD producing 3′-tailed ssDNA, onto which the RecA protein is preferentially loaded, thereby facilitating the initiation step of homologous recombination. In addition to controlling the polarity of the RecBCD nuclease activity, the encounter with χ results in an uncoupling of the two RecBCD helicase motors (RecB and RecD), so that DNA unwinding beyond χ is powered by the slower RecB helicase with the RecD motor having been inactivated at χ. Thus, a properly oriented χ-sequence controls the polarity of degradation and the rate of enzyme translocation. To understand more clearly how these activities are combined in the heterotrimeric enzyme and to understand more clearly how DNA alkylation might effect DNA helicase activity, we summarize the relevant features of the structure of RecBCD determined in the presence of DNA. The RecB and RecC subunits are intimately associated with one another, with the RecD subunit being more peripheral (Figure 2(a)). The RecB and RecD subunits couple the hydrolysis of ATP to DNA translocation and strand separation by pulling DNA into the holoenzyme, and through the RecC subunit, where strand separation occurs., Although RecC has no demonstrable enzymatic activity, it acts as a scaffold onto which the two motors assemble, it is intimately involved in strand separation and is responsible for χ recognition., Finally, the enzyme contains a single nuclease active site that resides within the C terminus of the RecB subunit, and which is positioned approximately opposite the entry point of dsDNA into the holoenzyme.