1887

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 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 strain was approximately twofold higher, whereas the mutation rate of a strain deficient in the family 1 UDG ( ) was found to be ∼8.4-fold higher. Interestingly, the mutation rate of the double-knockout ( / ) strain was remarkably high, at ∼19.6-fold. While CG to TA mutations predominated in the and / strains, AT to GC mutations were enhanced in the strain. The / strain was notably more sensitive to acidified nitrite and hydrogen peroxide stresses compared with the single knockouts ( or ). These observations reveal a synergistic effect of UdgB and Ung in DNA repair, and could have implications for the generation of attenuated strains of .

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.034363-0
2010-03-01
2021-03-08
Loading full text...

Full text loading...

/deliver/fulltext/micro/156/3/940.html?itemId=/content/journal/micro/10.1099/mic.0.034363-0&mimeType=html&fmt=ahah

References

  1. Acharya N., Kumar P., Varshney U. 2003; Complexes of the uracil-DNA glycosylase inhibitor protein, Ugi, with Mycobacterium smegmatis and Mycobacterium tuberculosis uracil-DNA glycosylases. Microbiology 149:1647–1658
    [Google Scholar]
  2. Bardarov S., Bardarov S. Jr, Pavelka M. S. Jr, Sambandamurthy V., Larsen M., Tufariello J., Chan J., Hatfull G., Jacobs W. R. Jr 2002; Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M.bovis BCG and M. smegmatis. Microbiology 148:3007–3017
    [Google Scholar]
  3. Boshoff H. I., Reed M. B., Barry C. E. III, Mizrahi V. 2003; DnaE2 polymerase contributes to in vivo survival and the emergence of drug resistance in Mycobacterium tuberculosis. Cell 113:183–193
    [Google Scholar]
  4. Cole S. T., Brosch R., Parkhill J., Garnier T., Churcher C., Harris D., Gordon S. V., Eiglmeier K., Gas S. other authors 1998; Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544
    [Google Scholar]
  5. David H. L. 1970; Probability distribution of drug-resistant mutants in unselected populations of Mycobacterium tuberculosis. Appl Microbiol 20:810–814
    [Google Scholar]
  6. Krokan H. E., Standal R., Slupphaug G. 1997; DNA glycosylases in the base excision repair of DNA. Biochem J 325:1–16
    [Google Scholar]
  7. Kurthkoti K., Kumar P., Jain R., Varshney U. 2008; Important role of the nucleotide excision repair pathway in Mycobacterium smegmatis in conferring protection against commonly encountered DNA-damaging agents. Microbiology 154:2776–2785
    [Google Scholar]
  8. Kurthkoti K., Srinath T., Kumar P., Malshetty V. S., Sang P. B., Jain R., Manjunath R., Varshney U. 2010; A distinct physiological role of MutY in mutation prevention in mycobacteria. Microbiology 156:88–98
    [Google Scholar]
  9. Lindahl T. 1993; Instability and decay of the primary structure of DNA. Nature 362:709–715
    [Google Scholar]
  10. Mizrahi V., Andersen S. J. 1998; DNA repair in Mycobacterium tuberculosis. What have we learnt from the genome sequence ?. Mol Microbiol 29:1331–1339
    [Google Scholar]
  11. Pelicic V., Jackson M., Reyrat J. M., Jacobs W. R. Jr, Gicquel B., Guilhot C. 1997; Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 94:10955–10960
    [Google Scholar]
  12. Rand L., Hinds J., Springer B., Sander P., Buxton R. S., Davis E. O. 2003; The majority of inducible DNA repair genes in Mycobacterium tuberculosis are induced independently of RecA. Mol Microbiol 50:1031–1042
    [Google Scholar]
  13. Reed K. C., Mann D. A. 1985; Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res 13:7207–7221
    [Google Scholar]
  14. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  15. Sartori A. A., Fitz-Gibbon S., Yang H., Miller J. H., Jiricny J. 2002; A novel uracil- DNA glycosylase with broad substrate specificity and an unusual active site. EMBO J 21:3182–3191
    [Google Scholar]
  16. Sassetti C. M., Rubin E. J. 2003; Genetic requirements for mycobacterial survival during infection. Proc Natl Acad Sci U S A 100:12989–12994
    [Google Scholar]
  17. Seshadri A., Samhita L., Gaur R., Malshetty V., Varshney U. 2009; Analysis of the fusA2 locus encoding EFG2 in Mycobacterium smegmatis. Tuberculosis (Edinb ) 89:453–464
    [Google Scholar]
  18. Snapper S. B., Melton R. E., Mustafa S., Kieser T., Jacobs W. R. Jr 1990; Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol 4:1911–1919
    [Google Scholar]
  19. Srinath T., Bharti S. K., Varshney U. 2007; Substrate specificities and functional characterization of a thermo-tolerant uracil DNA glycosylase (UdgB) from Mycobacterium tuberculosis. DNA Repair (Amst ) 6:1517–1528
    [Google Scholar]
  20. Starkuviene V., Fritz H. J. 2002; A novel type of uracil-DNA glycosylase mediating repair of hydrolytic DNA damage in the extremely thermophilic eubacterium Thermus thermophilus. Nucleic Acids Res 30:2097–2102
    [Google Scholar]
  21. Varshney U., van de Sande J. H. 1991; Specificities and kinetics of uracil excision from uracil-containing DNA oligomers by Escherichia coli uracil DNA glycosylase. Biochemistry 30:4055–4061
    [Google Scholar]
  22. Vasanthakrishna M., Kumar N. V., Varshney U. 1997; Characterization of the initiator tRNA gene locus and identification of a strong promoter from Mycobacterium tuberculosis. Microbiology 143:3591–3598
    [Google Scholar]
  23. Venkatesh J., Kumar P., Krishna P. S., Manjunath R., Varshney U. 2003; Importance of uracil DNA glycosylase in Pseudomonas aeruginosa and Mycobacterium smegmatis, G+C-rich bacteria, in mutation prevention, tolerance to acidified nitrite, and endurance in mouse macrophages. J Biol Chem 278:24350–24358
    [Google Scholar]
  24. Wanner R. M., Castor D., Guthlein C., Bottger E. C., Springer B., Jiriciny J. 2009; Uracil DNA glycosylase UdgB of Mycobacterium smegmatis protects the organism from the mutagenic effects of cytosine and adenine deamination. J Bacteriol 191:6312–6319
    [Google Scholar]
  25. Wink D. A., Kasprzak K. S., Maragos C. M., Elespuru R. K., Misra M., Dunams T. M., Cebula T. A., Koch W. H., Andrews A. W. other authors 1991; DNA deaminating ability and genotoxicity of nitric oxide and its progenitors. Science 254:1001–1003
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.034363-0
Loading
/content/journal/micro/10.1099/mic.0.034363-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error