RecD2 helicase Limits Replication Fork Stress in Bacillus subtilis.

Abstract

DNA helicases have important roles in replication, recombination, and repair. RecD2 has been identified for study by homology to the Escherichia coli RecD helicase. E. coli RecD has been well studied as a component of the RecBCD helicase-nuclease enzyme important for double-strand break repair. Bacillus subtilis and many other bacteria lack RecB and RecC and instead contain a RecD2 helicase, which is not known to function as part of a larger complex. Depending on the organism, RecD2 helicases have been shown to provide resistance to a broad range of DNA damaging agents while also contributing to the mismatch repair pathway. I have investigated the importance of B. subtilis RecD2 to genome integrity. Biochemical characterization of RecD2 indicates it is a 5´ to 3´ helicase and that it directly binds single-stranded DNA binding protein. I found that loss of RecD2 sensitized cells to a variety of DNA damaging agents including mitomycin C and the DNA break-inducing peptide phleomycin. I showed that deletion or overexpression of recD2 conferred a modest increase in spontaneous mutagenesis and induced the SOS response, and overexpression resulted in cell death. Further characterization included measurement of replication fork progression in vivo and showed that fork movement was slowed in ∆recD2 cells as well as during recD2 overexpression, supporting the hypothesis that normal levels of RecD2 are important for efficient replication fork movement. I identified specific variants of RecD2 that mitigate ectopic expression-induced toxicity while also conferring sensitivity to DNA damaging agents at low levels of expression. Together, these results highlight novel roles for RecD2 in DNA replication, by helping to maintain replication fork integrity during normal growth and following DNA damage, while also demonstrating that dysregulation of RecD2 is toxic.