Doug Julin
Associate Professor of Biochemistry
Department of Chemistry and Biochemistry
University of Maryland

 The RecBCD enzyme found in E. coli and many other bacteria functions in the recombinational repair of double-strand DNA breaks. These occur frequently during DNA replication and are lethal if unrepaired. RecBCD is a complex enzyme with both DNA helicase (DNA unwinding) and nuclease activities. The nuclease is regulated by a specific sequence called Chi (5'-GCTGGTGG) in double-stranded DNA.

RecBCD is made up of three protein subunits - called RecB, RecC, and RecD. We have been interested for some time in the enzymatic functions of these subunits. Amino acid sequence comparisons provided some clues - the RecB and RecD proteins are each similar to a large family of DNA helicases. The isolated RecB and RecD proteins are both DNA-dependent ATPases and weak helicases and the RecB helicase activity is essential for RecBCD function. We showed that RecB is also a nuclease and that the protein is constructed of two independent domains: an N-terminal helicase domain linked to a C-terminal nuclease domain. The function of RecD in RecBCD remains somewhat mysterious. Its helicase activity is not essential for RecBCD function since RecBC (no RecD subunit) is a very efficient DNA helicase. However, RecBC has greatly reduced nuclease activity compared to RecBCD. No nuclease is detectable in the isolated RecD protein and its function in the nuclease activity is unknown.

We are currently interested in the following issues, for which sequence analysis is providing interesting information.

1. The recB gene presumably arose by fusion of genes encoding a helicase and a nuclease. The helicase domain in RecB is closely related to that in many other helicases, including UvrD and Rep from bacteria. We are curious whether the nuclease domain of RecB is found in any other nucleases.

2. Some bacteria (e.g., Bacillus subtilis) do not have a RecBCD enzyme. Instead, they have a two-subunit enzyme (called AddAB in B. subtilis) that has the same enzymatic activities as RecBCD and serves the same biological function. The AddA subunit is similar to RecB, and the AddB subunit is thought to have arisen from a gene duplication of AddA. There is no evidence for a third subunit analogous to RecD in E. coli and other bacteria.

3. The RecD helicase domain is only distantly related to RecB, UvrD, and Rep. Analysis of several completely sequenced bacterial genomes reveals an interesting family of RecD homologues. We first became aware of these after the annotation of the genome of Deinococcus radiodurans, an fascinating bacterium that can withstand extremely high levels of ionizing radiation and other DNA-damaging agents. Interestingly, this organism was found to encode a RecD protein but neither RecB nor RecC. Further analysis suggests that this "RecD" protein is found in other bacteria as well, including several that have the AddAB type helicase/nuclease enzyme (B. subtilis and others). Our current work is on isolating and characterizing proteins that are related to the nuclease domain of RecB and this new family of RecD-like proteins.