Characterization of Three Mycobacterial DinB (DNA Polymerase IV) Paralogs Highlights DinB2 As Naturally Adept at Ribonucleotide Incorporation

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2014 Sep 10

PMID 25200080

Citations 27

Authors

Heather Ordonez

Maria Loressa Uson

Stewart Shuman

Affiliations

Soon will be listed here.

Abstract

This study unveils Mycobacterium smegmatis DinB2 as the founder of a clade of Y-family DNA polymerase that is naturally adept at incorporating ribonucleotides by virtue of a leucine in lieu of a canonical aromatic steric gate. DinB2 efficiently scavenges limiting dNTP and rNTP substrates in the presence of manganese. DinB2's sugar selectivity factor, gauged by rates of manganese-dependent dNMP versus rNMP addition, is 2.7- to 3.8-fold. DinB2 embeds ribonucleotides during DNA synthesis when rCTP and dCTP are at equimolar concentration. DinB2 can incorporate at least 16 consecutive ribonucleotides. In magnesium, DinB2 has a 26- to 78-fold lower affinity for rNTPs than dNTPs, but only a 2.6- to 6-fold differential in rates of deoxy versus ribo addition (kpol). Two other M. smegmatis Y-family polymerases, DinB1 and DinB3, are characterized here as template-dependent DNA polymerases that discriminate strongly against ribonucleotides, a property that, in the case of DinB1, correlates with its aromatic steric gate side chain. We speculate that the unique ability of DinB2 to utilize rNTPs might allow for DNA repair with a 'ribo patch' when dNTPs are limiting. Phylogenetic analysis reveals DinB2-like polymerases, with leucine, isoleucine or valine steric gates, in many taxa of the phylum Actinobacteria.

Citing Articles

A nucleoid-associated protein is involved in the emergence of antibiotic resistance by promoting the frequent exchange of the replicative DNA polymerase in .

Ng W, Rego E mSphere. 2024; 9(5):e0012224.

PMID: 38591887 PMC: 11237743. DOI: 10.1128/msphere.00122-24.

A nucleoid-associated protein is involved in the emergence of antibiotic resistance by promoting the frequent exchange of the replicative DNA polymerase in .

Ng W, Rego E bioRxiv. 2024; .

PMID: 38260554 PMC: 10802252. DOI: 10.1101/2023.06.12.544663.

Processing of matched and mismatched rNMPs in DNA by archaeal ribonucleotide excision repair.

Reveil M, Chapel L, Vourch B, Bosse A, Vialle L, Brizard R iScience. 2023; 26(12):108479.

PMID: 38077150 PMC: 10698281. DOI: 10.1016/j.isci.2023.108479.

Roles for mycobacterial DinB2 in frameshift and substitution mutagenesis.

Dupuy P, Ghosh S, Fay A, Adefisayo O, Gupta R, Shuman S Elife. 2023; 12.

PMID: 37141254 PMC: 10159617. DOI: 10.7554/eLife.83094.

The C-Terminal Acid Phosphatase Module of the RNase HI Enzyme RnhC Controls Rifampin Sensitivity and Light-Dependent Colony Pigmentation of Mycobacterium smegmatis.

Dupuy P, Glickman M J Bacteriol. 2023; 205(4):e0043122.

PMID: 36916909 PMC: 10127661. DOI: 10.1128/jb.00431-22.

References

Brissett N, Pitcher R, Juarez R, Picher A, Green A, Dafforn T . Structure of a NHEJ polymerase-mediated DNA synaptic complex. Science. 2007; 318(5849):456-9. DOI: 10.1126/science.1145112. View

Ordonez H, Unciuleac M, Shuman S . Mycobacterium smegmatis RqlH defines a novel clade of bacterial RecQ-like DNA helicases with ATP-dependent 3'-5' translocase and duplex unwinding activities. Nucleic Acids Res. 2012; 40(10):4604-14. PMC: 3378886. DOI: 10.1093/nar/gks046. View

Warner D, Ndwandwe D, Abrahams G, Kana B, Machowski E, Venclovas C . Essential roles for imuA'- and imuB-encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A. 2010; 107(29):13093-8. PMC: 2919956. DOI: 10.1073/pnas.1002614107. View

Yakovleva L, Shuman S . Mycobacterium smegmatis SftH exemplifies a distinctive clade of superfamily II DNA-dependent ATPases with 3' to 5' translocase and helicase activities. Nucleic Acids Res. 2012; 40(15):7465-75. PMC: 3424565. DOI: 10.1093/nar/gks417. View

Akey D, Martins A, Aniukwu J, Glickman M, Shuman S, Berger J . Crystal structure and nonhomologous end-joining function of the ligase component of Mycobacterium DNA ligase D. J Biol Chem. 2006; 281(19):13412-13423. DOI: 10.1074/jbc.M513550200. View

Sharma A, Kottur J, Narayanan N, Nair D . A strategically located serine residue is critical for the mutator activity of DNA polymerase IV from Escherichia coli. Nucleic Acids Res. 2013; 41(9):5104-14. PMC: 3643571. DOI: 10.1093/nar/gkt146. View

Yang G, Franklin M, Li J, Lin T, Konigsberg W . A conserved Tyr residue is required for sugar selectivity in a Pol alpha DNA polymerase. Biochemistry. 2002; 41(32):10256-61. DOI: 10.1021/bi0202171. View

Gong C, Martins A, Bongiorno P, Glickman M, Shuman S . Biochemical and genetic analysis of the four DNA ligases of mycobacteria. J Biol Chem. 2004; 279(20):20594-606. DOI: 10.1074/jbc.M401841200. View

Sinha K, Stephanou N, Gao F, Glickman M, Shuman S . Mycobacterial UvrD1 is a Ku-dependent DNA helicase that plays a role in multiple DNA repair events, including double-strand break repair. J Biol Chem. 2007; 282(20):15114-25. DOI: 10.1074/jbc.M701167200. View

10.

Sinha K, Unciuleac M, Glickman M, Shuman S . AdnAB: a new DSB-resecting motor-nuclease from mycobacteria. Genes Dev. 2009; 23(12):1423-37. PMC: 2701575. DOI: 10.1101/gad.1805709. View

11.

Zhu H, Bhattarai H, Yan H, Shuman S, Glickman M . Characterization of Mycobacterium smegmatis PolD2 and PolD1 as RNA/DNA polymerases homologous to the POL domain of bacterial DNA ligase D. Biochemistry. 2012; 51(51):10147-58. PMC: 3766730. DOI: 10.1021/bi301202e. View

12.

Kana B, Abrahams G, Sung N, Warner D, Gordhan B, Machowski E . Role of the DinB homologs Rv1537 and Rv3056 in Mycobacterium tuberculosis. J Bacteriol. 2010; 192(8):2220-7. PMC: 2849458. DOI: 10.1128/JB.01135-09. View

13.

Aniukwu J, Glickman M, Shuman S . The pathways and outcomes of mycobacterial NHEJ depend on the structure of the broken DNA ends. Genes Dev. 2008; 22(4):512-27. PMC: 2238672. DOI: 10.1101/gad.1631908. View

14.

Bjedov I, Dasgupta C, Slade D, Le Blastier S, Selva M, Matic I . Involvement of Escherichia coli DNA polymerase IV in tolerance of cytotoxic alkylating DNA lesions in vivo. Genetics. 2007; 176(3):1431-40. PMC: 1931539. DOI: 10.1534/genetics.107.072405. View

15.

Boshoff H, Reed M, Barry 3rd C, Mizrahi V . DnaE2 polymerase contributes to in vivo survival and the emergence of drug resistance in Mycobacterium tuberculosis. Cell. 2003; 113(2):183-93. DOI: 10.1016/s0092-8674(03)00270-8. View

16.

Brissett N, Martin M, Pitcher R, Bianchi J, Juarez R, Green A . Structure of a preternary complex involving a prokaryotic NHEJ DNA polymerase. Mol Cell. 2011; 41(2):221-31. DOI: 10.1016/j.molcel.2010.12.026. View

17.

Courcelle J, Khodursky A, Peter B, Brown P, HANAWALT P . Comparative gene expression profiles following UV exposure in wild-type and SOS-deficient Escherichia coli. Genetics. 2001; 158(1):41-64. PMC: 1461638. DOI: 10.1093/genetics/158.1.41. View

18.

Sinha K, Stephanou N, Unciuleac M, Glickman M, Shuman S . Domain requirements for DNA unwinding by mycobacterial UvrD2, an essential DNA helicase. Biochemistry. 2008; 47(36):9355-64. PMC: 2648833. DOI: 10.1021/bi800725q. View

19.

Shuman S, Glickman M . Bacterial DNA repair by non-homologous end joining. Nat Rev Microbiol. 2007; 5(11):852-61. DOI: 10.1038/nrmicro1768. View

20.

Nick McElhinny S, Watts B, Kumar D, Watt D, Lundstrom E, Burgers P . Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases. Proc Natl Acad Sci U S A. 2010; 107(11):4949-54. PMC: 2841928. DOI: 10.1073/pnas.0914857107. View