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J. Biol. Chem., Vol. 278, Issue 25, 22446-22452, June 20, 2003

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DNA Loop Repair by Escherichia coli Cell Extracts^*

Woei-horng Fang   ¶, Bo-Jeng Wang , Chiang-Hui Wang , Su-Jan Lee , Yu-Ting Chang , Yi-Kuang Chuang  and Chun-Nan Lee  

From the School of Medical Technology, College of Medicine, National Taiwan University and the Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan 100, Republic of China

The nick-directed DNA repair efficiency of a set of M13mp18-derived heteroduplexes containing 8-, 12-, 16-, 22-, 27-, 45-, and 429-nucleotide loops was determined by in vitro assay. Unpaired nucleotides of each heteroduplex reside within overlapping recognition sites for two restriction endonucleases, permitting independent evaluation of repair occurring on either DNA strand. Our results show that a strand break located either 3' or 5' to the loop is sufficient to direct heterology repair to the nicked strand in Escherichia coli extracts. Strand-specific repair by this system requires Mg²⁺ and the four dNTPs and is equally efficient on insertions and deletions. This activity is distinct from the MutHLS mismatch repair pathway. Strand specificity and repair efficiency are largely independent of the GATC methylation state of the DNA and presence of the products of mismatch repair genes mutH, mutL, and mutS. This study provides evidence for a loop repair pathway in E. coli that is distinct from conventional mismatch repair.

Received for publication, March 13, 2003 , and in revised form, April 9, 2003.

^* This work was supported by National Science Council (Taipei, Taiwan, Republic of China) Grant NSC 89-2320-B-002-093. 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.

^¶ To whom correspondence should be addressed. Tel.: 886-223123456 (ext. 6926); Fax: 886-2-23711574; E-mail: whfang{at}ha.mc.ntu.edu.tw.

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