Synergistic effects of UdgB and Ung in mutation prevention and protection against commonly encountered DNA damaging agents in Mycobacterium smegmatis

Abstract

The incorporation of dUMP during replication or the deamination of cytosine in DNA results in the occurrence of uracils in genomes. To maintain genomic integrity, uracil DNA glycosylases (UDGs) excise uracil from DNA and initiate the base-excision repair pathway. Here, we cloned, purified and biochemically characterized a family 5 UDG, UdgB, from Mycobacterium smegmatis to allow us to use it as a model organism to investigate the physiological significance of the novel enzyme. Studies with knockout strains showed that compared with the wild-type parent, the mutation rate of the udgB₋ strain was approximately twofold higher, whereas the mutation rate of a strain deficient in the family 1 UDG (ung₋) was found to be ~8.4-fold higher. Interestingly, the mutation rate of the double-knockout (ung⁻/udgB⁻) strain was remarkably high, at ~19.6-fold. While CG to TA mutations predominated in the ung⁻ and ung⁻/udgB⁻ strains, AT to GC mutations were enhanced in the udgB⁻ strain. The ung⁻/udgB⁻ strain was notably more sensitive to acidified nitrite and hydrogen peroxide stresses compared with the single knockouts (ung⁻ or udgB⁻). These observations reveal a synergistic effect of UdgB and Ung in DNA repair, and could have implications for the generation of attenuated strains of Mycobacterium tuberculosis.