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Nucleic Acids Research Advance Access published online on July 15, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn446
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

Partial reconstitution of DNA large loop repair with purified proteins from Saccharomyces cerevisiae

Debbie Sommer1, Carrie M. Stith2, Peter M. J. Burgers2 and Robert S. Lahue1,3,*

1Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198-6805, 2Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO 63110, USA and 3Department of Biochemistry, National University of Ireland, Galway, Ireland

*To whom correspondence should be addressed. Tel: +353 91 495756; Fax: +353 91 495504; Email: bob.lahue{at}nuigalway.ie

Received May 12, 2008. Revised June 23, 2008. Accepted June 26, 2008.

Small looped mispairs are corrected by DNA mismatch repair. In addition, a distinct process called large loop repair (LLR) corrects heteroduplexes up to several hundred nucleotides in bacteria, yeast and human cells, and in cell-free extracts. Only some LLR protein components are known, however. Previous studies with neutralizing antibodies suggested a role for yeast DNA polymerase {delta} (Pol {delta}), RFC and PCNA in LLR repair synthesis. In the current study, biochemical fractionation studies identified FEN1 (Rad27) as another required LLR component. In the presence of purified FEN1, Pol {delta}, RFC and PCNA, repair occurred on heteroduplexes with loops ranging from 8 to 216 nt. Repair utilized a 5' nick, with correction directed to the nicked strand, irrespective of which strand contained the loop. In contrast, repair of a G/T mismatch occurred at low levels, suggesting specificity of the reconstituted system for looped mispairs. The presence of RPA enhanced reactivity on some looped substrates, but RPA was not required for activity. Although additional LLR factors remain to be identified, the excision and resynthesis steps of LLR from a 5' nick can be reconstituted in a purified system with FEN1 and Pol {delta}, together with PCNA and its loader RFC.


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