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Originally published In Press as doi:10.1074/jbc.M603302200 on June 30, 2006

J. Biol. Chem., Vol. 281, Issue 35, 25026-25040, September 1, 2006
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Nucleotide Misincorporation, 3'-Mismatch Extension, and Responses to Abasic Sites and DNA Adducts by the Polymerase Component of Bacterial DNA Ligase D*

Lyudmila Yakovleva and Stewart Shuman1

From the Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021

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.


Received for publication, April 6, 2006 , and in revised form, June 28, 2006.

* This work was supported in part by National Institutes of Health Grant GM63611. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 American Cancer Society Research Professor. To whom correspondence should be addressed. E-mail: s-shuman{at}ski.mskcc.org.


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