Synergistic effects of UdgB and Ung in mutation prevention and protection against commonly encountered DNA damaging agents in Mycobacterium smegmatis.
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 approximately 8.4-fold higher. Interestingly, the mutation rate of the double-knockout (ung(-)/ udgB(-)) strain was remarkably high, at approximately 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.
Malshetty
Vidyasagar S
VS
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Jain
Ruchi
R
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Srinath
Thiruneelakantan
T
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Kurthkoti
Krishna
K
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Varshney
Umesh
U
Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, 560064, India.|Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004249:DNA Damage; D004260:DNA Repair; D004269:DNA, Bacterial / Q000378:metabolism*; D055786:Gene Knockout Techniques; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D016415:Sequence Alignment; D012977:Sodium Nitrite / Q000494:pharmacology; D051981:Uracil-DNA Glycosidase / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006861:Hydrogen Peroxide; D051981:Uracil-DNA Glycosidase; D012977:Sodium Nitrite