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Molecular and Cellular Biology, November 2009, p. 5998-6005, Vol. 29, No. 22
0270-7306/09/$08.00+0     doi:10.1128/MCB.01127-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Meiotic DNA Double-Strand Break Repair Requires Two Nucleases, MRN and Ctp1, To Produce a Single Size Class of Rec12 (Spo11)-Oligonucleotide Complexes{triangledown}

Neta Milman, Emily Higuchi, and Gerald R. Smith*

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109

Received 19 August 2009/ Returned for modification 26 August 2009/ Accepted 1 September 2009

Programmed DNA double-strand breaks (DSBs) in meiosis are formed by Spo11 (Rec12 in fission yeast), a topoisomerase II-like protein, which becomes covalently attached to DNA 5' ends. For DSB repair through homologous recombination, the protein must be removed from these DNA ends. We show here that Rec12 is endonucleolytically removed from DSB ends attached to a short oligonucleotide (Rec12-oligonucleotide complex), as is Spo11 in budding yeast. Fission yeast, however, has only one size class of Rec12-oligonucleotide complexes, whereas budding yeast has two size classes, suggesting different endonucleolytic regulatory mechanisms. Rec12-oligonucleotide generation strictly requires Ctp1 (Sae2 nuclease homolog), the Rad32 (Mre11) nuclease domain, and Rad50 of the MRN complex. Surprisingly, Nbs1 is not strictly required, indicating separable roles for the MRN subunits. On the basis of these and other data, we propose that Rad32 nuclease has the catalytic site for Rec12-oligonucleotide generation and is activated by Ctp1, which plays an additional role in meiotic recombination.

* Corresponding author. Mailing address: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, A1-162, Seattle, WA 98109. Phone: (206) 667-4438. Fax: (206) 667-6497. E-mail: gsmith{at}fhcrc.org

{triangledown} Published ahead of print on 14 September 2009.

Molecular and Cellular Biology, November 2009, p. 5998-6005, Vol. 29, No. 22
0270-7306/09/$08.00+0     doi:10.1128/MCB.01127-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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