The RecBCD enzyme of Escherichia coli is an ATP-dependent DNA exonuclease and a helicase. Its exonuclease activity is subject to regulation by an octameric nucleotide sequence called chi. In this study, site-directed mutations were made in the carboxyl-terminal nuclease domain of the RecB subunit, and their effects on RecBCD's enzymatic activities were investigated. Mutation of two amino acid residues, Asp(1067) and Lys(1082), abolished nuclease activity on both single- and double-stranded DNA. Together with Asp(1080), these residues compose a motif that is similar to one shown to form the active site of several restriction endonucleases. The nuclease reactions catalyzed by the RecBCD enzyme should therefore follow the same mechanism as these restriction endonucleases. Furthermore, the mutant enzymes were unable to produce chi-specific fragments that are thought to result from the 3'-5' and 5'-3' single-stranded exonuclease activities of the enzyme during its reaction with chi-containing double-stranded DNA. The results show that the nuclease active site in the RecB C-terminal 30-kDa domain is the universal nuclease active site of RecBCD that is responsible for DNA degradation in both directions during the reaction with double-stranded DNA. A novel explanation for the observed nuclease polarity switch and RecBCD-DNA interaction is offered.

We have expressed the RecD subunit of the RecBCD enzyme from Escherichia coli as a fusion protein with a 31-amino acid NH 2 -terminal extension including 6 consecutive histidine residues (HisRecD). The overexpressed fusion protein can be purified in urea-denatured form by metal chelate affinity chromatography. The mixture of renatured HisRecD protein and the RecB and RecC proteins has a high level of ATP-dependent nuclease activity with either single-or double-stranded DNA, enhanced DNA unwinding activity, enhanced ATP hydrolysis activity in the presence of a small DNA oligomer cosubstrate, and -cutting activity. These are all characteristics of the RecBCD holoenzyme. The HisRecD protein by itself hydrolyzes ATP in the presence of high concentrations of single-stranded DNA (polydeoxythymidine). The activity is unstable at 37°C, but is measurable at room temperature (about 23°C). The HisRecD has very little ATPase activity in the presence of a much shorter single-stranded DNA (oligodeoxy(thymidine) 12 ). HisRecD hydrolyzes ATP more efficiently than GTP and UTP, and has very little activity with CTP. We also purified a fusion protein containing a Lys to Gln mutation in the putative ATP-binding site of RecD. This mutant protein has no ATPase activity, indicating that the observed ATP hydrolysis activity is intrinsic to the RecD protein itself.The RecBCD enzyme from Escherichia coli is an important enzyme in the DNA metabolism of the cell, acting in homologous recombination, resistance to UV irradiation and chemical DNA damaging agents, and degradation of foreign DNA (reviewed in Refs. 1-3). The enzyme catalyzes several reactions in vitro. ATP hydrolysis enables it to unwind double-stranded DNA (4 -6), and the enzyme is a potent nuclease on double-stranded DNA in the presence of ATP and excess magnesium ion (7,8). The doublestrand nuclease activity of RecBCD is suppressed when it encounters a sequence (5Ј-GCTGGTGG) in the DNA (9 -12). The enzyme continues to unwind the DNA past the sequence (11) and the unwound DNA is a substrate for recombination catalyzed by the RecA protein (13-15). RecBCD enzyme is also an ATPstimulated nuclease with single-stranded DNA, and singlestranded DNA stimulates ATP hydrolysis (7,8).The first preparations of the RecBCD enzyme were thought to contain only the proteins encoded by the recB and recC genes (8, 16 -19). However, one experiment indicated the possible existence of an additional component, essential for enzymatic activity, which could be separated from both the RecB and RecC proteins by treating the enzyme with high salt concentrations (20). A later study showed that this was a third protein subunit, called RecD, encoded by a gene (recD) adjacent to the recB gene (21).The function of the RecD subunit in catalysis by RecBCD enzyme is not clear, although it is implicated in several activities of the enzyme. The identification of the RecD subunit was made possible by the fact that it is required for high levels of nuclease activity with single-or double-stranded DNA (21,22). Amin...

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