BIOLOGICAL CHARACTERIZATION OF RECD MUTANTS IN DEINOCOCCUS RADIODURANS

Abstract

The RecD protein in E. coli and other gram negative bacteria functions as a subunit in concert with RecB and RecC to form the heterotrimeric RecBCD complex. It is involved in degrading foreign linear DNA, providing a block against viral invaders, and has been shown to inhibit recombination. This complex also catalyzes the first step in the highly regulated enzymatic process of DSBR via homologous recombination (HR). DSBR via HR is thought to be the major route for processing double strand breaks in E. coli. The first step is known as initiation and generates the substrate for the next enzyme in the pathway, RecA. In contrast to E. coli, most gram positive bacteria, use a set of genes known as the AddAB genes to accomplish this same task, to produce the substrate for RecA. The gram positive bacterium Deinococcus radiodurans is remarkable in its ability to withstand DNA damaging agents such as MMC, gamma irradiation, hydrogen peroxide and UV irradiation. While D. radiodurans has a RecA homolog that has been shown to be critical for repair of double strand breaks, sequence analysis has not revealed homologs of AddAB or RecBC. A sequence with high similarity to recD was found. D. radiodurans RecD has an extended N-terminus grouping it with a sub-family of recD-like genes. The gene was expressed in E. coli and purified. Its biochemical activities were published by Wang and Julin, 2004, showing it had 5'-3' helicase activity like RecD proteins from other organisms. In this study a recD mutation in D. radiodurans was generated. The mutant was sensitive to gamma and UV irradiation, and hydrogen peroxide. The mutant was insensitive to MMC and MMS. Additionally the mutant showed an increased capacity to be transformed by exogenous DNA. These results imply a role in DNA repair through some form of recombinational repair. This finding is the first published result of a member of the recD-like sub-family of proteins that suggests role in DNA repair.