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J. Biol. Chem., Vol. 283, Issue 34, 23343-23352, August 22, 2008

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Structure-guided Mutational Analysis of the OB, HhH, and BRCT Domains of Escherichia coli DNA Ligase^*

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

NAD⁺-dependent DNA ligases (LigAs) are ubiquitous in bacteria and essential for growth. LigA enzymes have a modular structure in which a central catalytic core composed of nucleotidyltransferase and oligonucleotide-binding (OB) domains is linked via a tetracysteine zinc finger to distal helix-hairpin-helix (HhH) and BRCT (BRCA1-like C-terminal) domains. The OB and HhH domains contribute prominently to the protein clamp formed by LigA around nicked duplex DNA. Here we conducted a structure-function analysis of the OB and HhH domains of Escherichia coli LigA by alanine scanning and conservative substitutions, entailing 43 mutations at 22 amino acids. We thereby identified essential functional groups in the OB domain that engage the DNA phosphodiester backbone flanking the nick (Arg³³³); penetrate the minor grove and distort the nick (Val³⁸³ and Ile³⁸⁴); or stabilize the OB fold (Arg³⁷⁹). The essential constituents of the HhH domain include: four glycines (Gly⁴⁵⁵, Gly⁴⁸⁹, Gly⁵²¹, Gly⁵⁵³), which bind the phosphate backbone across the minor groove at the outer margins of the LigA-DNA interface; Arg⁴⁸⁷, which penetrates the minor groove at the outer margin on the 3 ®-OH side of the nick; and Arg⁴⁴⁶, which promotes protein clamp formation via contacts to the nucleotidyltransferase domain. We find that the BRCT domain is required in its entirety for effective nick sealing and AMP-dependent supercoil relaxation.

Received for publication, April 17, 2008 , and in revised form, May 26, 2008.

^* This work was supported, in whole or 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.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure.

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

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