Characterization of mycobacterium smegmatis pold2 and pold1 as RNA/DNA polymerases homologous to the POL domain of bacterial DNA ligase D Journal Article
| Authors: | Zhu, H.; Bhattarai, H.; Yan, H. G.; Shuman, S.; Glickman, M. S. |
| Article Title: | Characterization of mycobacterium smegmatis pold2 and pold1 as RNA/DNA polymerases homologous to the POL domain of bacterial DNA ligase D |
| Abstract: | Mycobacteria exploit nonhomologous end-joining (NHEJ) to repair DNA double-strand breaks. The core NHEJ machinery comprises the homodimeric DNA end-binding protein Ku and DNA ligase D (LigD), a modular enzyme composed of a C-terminal ATP-dependent ligase domain (LIG), a central 3′-phosphoesterase domain (PE), and an N-terminal polymerase domain (POL). LigD POL is proficient at adding templated and nontemplated deoxynucleotides and ribonucleotides to DNA ends in vitro and is the catalyst in vivo of unfaithful NHEJ events involving nontemplated single-nucleotide additions to blunt DSB ends. Here, we identify two mycobacterial proteins, PolD1 and PolD2, as stand-alone homologues of the LigD POL domain. Biochemical characterization of PolD1 and PolD2 shows that they resemble LigD POL in their monomeric quaternary structures, their ability to add templated and nontemplated nucleotides to primer-templates and blunt ends, and their preference for rNTPs versus dNTPs. Deletion of polD1, polD2, or both from a Mycobacterium smegmatis strain carrying an inactivating mutation in LigD POL failed to reveal a role for PolD1 or PolD2 in templated nucleotide additions during NHEJ of 5′-overhang DSBs or in clastogen resistance. Whereas our results document the existence and characteristics of new stand-alone members of the LigD POL family of RNA/DNA polymerases, they imply that other polymerases can perform fill-in synthesis during mycobacterial NHEJ. © 2012 American Chemical Society. |
| Keywords: | controlled study; unclassified drug; gene mutation; gene deletion; nonhuman; protein domain; proteins; phosphatase; carboxy terminal sequence; in vitro study; antibiotic resistance; dna; amino terminal sequence; protein synthesis; in-vivo; adenosine triphosphate; polydeoxyribonucleotide synthase; in-vitro; biochemistry; bacterial dna; machinery; dna ligases; dna directed rna polymerase; mycobacterial; mycobacterium smegmatis; double-strand breaks; ribonucleotide; nucleotides; polymers; bacteria; n-terminals; biochemical characterization; nonhomologous end joining; ribonucleotides; templated; deoxyribonucleotide; protein quaternary structure; modular enzymes; quaternary structure; clastogen; pold1 protein; pold2 protein |
| Journal Title: | Biochemistry |
| Volume: | 51 |
| Issue: | 51 |
| ISSN: | 0006-2960 |
| Publisher: | American Chemical Society |
| Date Published: | 2012-12-21 |
| Start Page: | 10147 |
| End Page: | 10158 |
| Language: | English |
| DOI: | 10.1021/bi301202e |
| PROVIDER: | scopus |
| PUBMED: | 23198659 |
| PMCID: | PMC3766730 |
| DOI/URL: | |
| Notes: | --- - "Export Date: 1 February 2013" - "CODEN: BICHA" - "Source: Scopus" |
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