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Molecular and Cellular Biology, February 2005, p. 896-906, Vol. 25, No. 3
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.3.896-906.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Rejoining of DNA Double-Strand Breaks as a Function of Overhang Length{dagger}

James M. Daley1 and Thomas E. Wilson2*

Department of Pathology,2 Graduate Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, Michigan1

Received 26 July 2004/ Returned for modification 31 August 2004/ Accepted 28 October 2004

The ends of spontaneously occurring double-strand breaks (DSBs) may contain various lengths of single-stranded DNA, blocking lesions, and gaps and flaps generated by end annealing. To investigate the processing of such structures, we developed an assay in which annealed oligonucleotides are ligated onto the ends of a linearized plasmid which is then transformed into Saccharomyces cerevisiae. Reconstitution of a marker occurs only when the oligonucleotides are incorporated and repair is in frame, permitting rapid analysis of complex DSB ends. Here, we created DSBs with compatible overhangs of various lengths and asked which pathways are required for their precise repair. Three mechanisms of rejoining were observed, regardless of overhang polarity: nonhomologous end joining (NHEJ), a Rad52-dependent single-strand annealing-like pathway, and a third mechanism independent of the first two mechanisms. DSBs with overhangs of less than 4 bases were mainly repaired by NHEJ. Repair became less dependent on NHEJ when the overhangs were longer or had a higher GC content. Repair of overhangs greater than 8 nucleotides was as much as 150-fold more efficient, impaired 10-fold by rad52 mutation, and highly accurate. Reducing the microhomology extent between long overhangs reduced their repair dramatically, to less than NHEJ of comparable short overhangs. These data support a model in which annealing energy is a primary determinant of the rejoining efficiency and mechanism.

* Corresponding author. Mailing address: Department of Pathology, University of Michigan Medical School, Medical Science I M4214/0602, 1301 Catherine Rd., Ann Arbor, MI 48109-0602. Phone: (734) 936-1887. Fax: (734) 763-6476. E-mail: wilsonte{at}umich.edu.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

Molecular and Cellular Biology, February 2005, p. 896-906, Vol. 25, No. 3
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.3.896-906.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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