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Nucleic Acids Research Advance Access originally published online on February 11, 2008
Nucleic Acids Research 2008 36(6):1836-1846; doi:10.1093/nar/gkm1148
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Nucleic Acids Research, 2008, Vol. 36, No. 6 1836-1846
© 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.

Molecular Biology

Apn1 and Apn2 endonucleases prevent accumulation of repair-associated DNA breaks in budding yeast as revealed by direct chromosomal analysis

Wenjian Ma, Michael A. Resnick and Dmitry A. Gordenin*

Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences (NIH, DHHS), Research Triangle Park, NC 27709, USA

*To whom correspondence should be addressed. Tel: +1 919 541 5190; Fax: +1 919 541 7593; Email: gordenin{at}niehs.nih.gov

Received October 12, 2007. Revised December 7, 2007. Accepted December 11, 2007.

Base excision repair (BER) provides relief from many DNA lesions. While BER enzymes have been characterized biochemically, BER functions within cells are much less understood, in part because replication bypass and double-strand break (DSB) repair can also impact resistance to base damage. To investigate BER in vivo, we examined the repair of methyl methanesulfonate (MMS) induced DNA damage in haploid G1 yeast cells, so that replication bypass and recombinational DSB repair cannot occur. Based on the heat-lability of MMS-induced base damage, an assay was developed that monitors secondary breaks in full-length yeast chromosomes where closely spaced breaks yield DSBs that are observed by pulsed-field gel electrophoresis. The assay detects damaged bases and abasic (AP) sites as heat-dependent breaks as well as intermediate heat-independent breaks that arise during BER. Using a circular chromosome, lesion frequency and repair kinetics could be easily determined. Monitoring BER in single and multiple glycosylase and AP-endonuclease mutants confirmed that Mag1 is the major enzyme that removes MMS-damaged bases. This approach provided direct physical evidence that Apn1 and Apn2 not only repair cellular base damage but also prevent break accumulation that can result from AP sites being channeled into other BER pathway(s).


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