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J. Biol. Chem., Vol. 283, Issue 13, 8331-8339, March 28, 2008

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Bacterial Nonhomologous End Joining Ligases Preferentially Seal Breaks with a 3'-OH Monoribonucleotide^*

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

Many bacterial species have a nonhomologous end joining system of DNA repair driven by dedicated DNA ligases (LigD and LigC). LigD is a multifunctional enzyme composed of a ligase domain fused to two other catalytic modules: a polymerase that preferentially adds ribonucleotides to double-strand break ends and a phosphoesterase that trims 3'-oligoribonucleotide tracts until only a single 3'-ribonucleotide remains. LigD and LigC have a feeble capacity to seal 3'-OH/5'-PO₄ DNA nicks. Here, we report that nick sealing by LigD and LigC enzymes is stimulated by the presence of a single ribonucleotide at the broken 3'-OH end. The ribonucleotide effect on LigD and LigC is specific for the 3'-terminal nucleotide and is either diminished or abolished when additional vicinal ribonucleotides are present. No such 3'-ribonucleotide effect is observed for bacterial LigA or Chlorella virus ligase. We found that in vitro repair of a double-strand break by Pseudomonas LigD requires the polymerase module and results in incorporation of an alkali-labile ribonucleotide at the repair junction. These results illuminate an underlying logic for the domain organization of LigD, whereby the polymerase and phosphoesterase domains can heal the broken 3'-end to generate the monoribonucleotide terminus favored by the nonhomologous end joining ligases.

Received for publication, July 5, 2007 , and in revised form, January 17, 2008.

^* This work was supported 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: Molecular Biology Program, Sloan-Kettering Inst., 1275 York Ave., New York, NY 10021. E-mail: s-shuman{at}ski.mskcc.org.

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