A Single Nuclease Active Site of the Escherichia coli RecBCD Enzyme Catalyzes Single-stranded DNA Degradation in Both Directions

Abstract

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.

High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides

“…Gam inhibits the activity of the RecBCD nuclease, as well as the hairpin nuclease SbcCD (19). Because RecBCD has single-stranded nuclease activity (20), the decreased recombination efficiency of ssDNA in a strain lacking Gam might be due to the RecBCD single-stranded nuclease activity, to the SbcCD hairpin nuclease activity, or perhaps to some unknown function normally inhibited by Gam. Beta bound to ssDNA protects it from nuclease attack (21); this may be another reason that ssDNA recombination is proficient even in the absence of Gam.…”

RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks

“…The N-terminal domain interacts with the RecC protein to form a rapid and processive DNA helicase (197). A C-terminal domain in RecB can function independently as a DNA endo-and exonuclease and is responsible for all nuclease activities associated with the RecBCD complex (278,308,324,325). The C-terminal nuclease domain of RecB was shown to interact directly with the RecA protein (67,270).…”

“…During DNA translocation and unwinding, RecBCD cleaves the DNA using a single nuclease active site (278,308,325,326). Extensive in vitro studies have established that the nuclease and translocation activities are independent (92,284,294).…”

“…Although named exonuclease V because the vigorous dsDNA degradation requires and initiates from a DNA end, from a mechanistic standpoint, the RecBCD enzyme is a nonspecific ssDNA endonuclease, and both the purified holoenzyme and the isolated RecB nuclease domain function as such in vitro (193,278,308,312). However, on linear duplex DNA substrates, the nuclease activity is manifest as an ATP-dependent dsDNA exonuclease due to the coupling of the endonucleolytic cleavage of ssDNA produced by DNA unwinding to the helicase activity (i.e., the helicase activity processively converts linear duplex DNA into an ssDNA substrate, which is concomitantly fed to the nuclease domain) (92,240,284,294).…”

Site-Directed Mutagenesis Using Oligonucleotide-Based Recombineering

“…Neither exo nor gam causes a dramatic effect on the recombination efficiency. Only a minor 5-fold reduction was observed in the gam deletion strain, most likely attributed to the single-stranded nuclease activity of the RecBCD protein complex, which is not inhibited in a gam-deficient strain , Wang et al, 2000. λ Red bound to ssDNA is able to protect the DNA segment from nuclease attack, which might explain the recombination events that occured despite the gam deletion .…”