Nucleotide Misincorporation, 3′-Mismatch Extension, and Responses to Abasic Sites and DNA Adducts by the Polymerase Component of Bacterial DNA Ligase D

Abstract

DNA ligase D (LigD) participates in a mutagenic pathway of nonhomologous end joining in bacteria. LigD consists of an ATP-dependent ligase domain fused to a polymerase domain (POL) and a phosphoesterase module. The POL domain performs templated and nontemplated primer extension reactions with either dNTP or rNTP substrates. Here we report that Pseudomonas LigD POL is an unfaithful nucleic acid polymerase. Although the degree of infidelity in nucleotide incorporation varies according to the mispair produced, we find that a correctly paired ribonucleotide is added to the DNA primer terminus more rapidly than the corresponding correct deoxyribonucleotide and incorrect nucleotides are added much more rapidly with rNTP substrates than with dNTPs, no matter what the mispair configuration. We find that 3 mispairs are extended by LigD POL, albeit more slowly than 3 paired primer-templates. The magnitude of the rate effect on mismatch extension varies with the identity of the 3 mispair, but it was generally the case that mispaired ends were extended more rapidly with rNTP substrates than with dNTPs. These results lend credence to the suggestion that LigD POL might fill in short 5-overhangs with ribonucleotides when repairing double strand breaks in quiescent cells. We report that LigD POL can add a deoxynucleotide opposite an abasic lesion in the template strand, albeit slowly. Ribonucleotides are inserted more rapidly at an abasic lesion than are deoxys. LigD POL displays feeble activity in extending a preformed primer terminus opposing an abasic site, but can readily bypass the lesion by slippage of the primer 3 di-or trinucleotide and realignment to the template sequence distal to the abasic site. Covalent benzo[a]pyrene-dG and benzo[c]phenanthrene-dA adducts in the template strand are durable roadblocks to POL elongation. POL can slowly insert a dNMP opposite the adduct, but is impaired in the subsequent extension step. Bacterial DNA ligase D (LigD)2 is distinguished from all other DNA ligases insofar as its enzymatic activity is not limited to sealing DNA strands. Rather, it is a polyfunctional repair enzyme consisting of an ATP-dependent ligase domain fused to a polymerase domain (POL) and a phosphoesterase domain (1-10). Interest in LigD is driven by evidence that it collaborates with a bacterial Ku homolog in a nonhomologous end joining (NHEJ) pathway characterized by a high incidence of frameshift mutations at the sites of double strand break (DSB) repair (1-4, 9, 10). Biochemical characterization of the POL and phosphoesterase components suggests that they provide a means of remodeling the 3Ј ends of broken DNA strands prior to sealing by the ligase component (2-10).The LigD POL domain catalyzes either nontemplated single nucleotide additions to a blunt-ended duplex DNA or fill-in synthesis at a 5Ј-tailed duplex DNA; these are the molecular signatures of mutagenic mycobacterial NHEJ in vivo at bluntend and 5Ј-overhang DSBs, respectively (2, 9, 10). POL activity in vitro is optimal in the presence of manganese (...

Biochemical Characterization of Mycobacterium smegmatis RnhC (MSMEG_4305), a Bifunctional Enzyme Composed of Autonomous N-Terminal Type I RNase H and C-Terminal Acid Phosphatase Domains

“…T he human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis have a large roster of DNA repair enzymes, including a shared set of eight DNA polymerases (1)(2)(3)(4)(5), four of which-LigD-POL, PolD1, PolD2, and DinB2-have the distinctive properties of low fidelity and readily incorporating ribonucleotides in lieu of deoxyribonucleotides during primer extension and gap repair in vitro (4)(5)(6)(7)(8)(9)(10). LigD-POL, PolD1, and PolD2 are paralogous members of the AEP (archaeal/eukaryal polymerase/primase) polymerase family (4,10,11).…”

The pathways and outcomes of mycobacterial NHEJ depend on the structure of the broken DNA ends

“…The POL and PE domains comprise a suite of DNA end-healing activities that are proposed to remodel the 3Ј terminus of the DSB prior to sealing by the LIG component. The POL domain is a minimized member of the archaeal-eukaryal primasepolymerase family that is especially adept at adding a short tract of ribonucleotides to a DNA primer-template and at adding a single nontemplated ribonucleotide at a blunt DNA end (Della et al 2004;Gong et al 2005;Zhu and Shuman 2005a;Yakovleva and Shuman 2006;Pitcher et al 2007c). The PE domain, which removes 3Ј phosphates and 3Ј ribonucleotides from primer-templates, exemplifies a new family of 3Ј processing enzymes with a distinctive substrate specificity and active site Shuman 2005b, 2006;Zhu et al 2005).…”

Bacterial Nonhomologous End Joining Ligases Preferentially Seal Breaks with a 3′-OH Monoribonucleotide

“…These are the molecular signatures of mutagenic mycobacterial NHEJ in vivo at blunt-end and 5Ј-overhang DSBs, respectively (1). Pseudomonas LigD POL uses manganese as a cofactor and strongly prefers rNTPs over dNTPs as substrates for both nontemplated blunt-end addition and templated fill-in synthesis (14,19). Templated ribonucleotide addition is limited to about four cycles of rNMP incorporation because the primer-template is rendered progressively less active as ribonucleotides accumulate at the 3Ј-end (14).…”