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Nucleic Acids Research Advance Access published online on August 15, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm552
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© 2007 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

Biochemical reconstitution of abasic DNA lesion replication in Xenopus extracts

Shuren Liao, Yoshihiro Matsumoto and Hong Yan*

Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA

*To whom correspondence should be addressed. Tel: +1 215 728 2514; Fax: +1 215 728 3574; Email: hong_yan{at}fccc.edu

Received March 28, 2007. Revised July 7, 2007. Accepted July 8, 2007.

Cellular DNA is under constant attack from numerous exogenous and endogenous agents. The resulting DNA lesions, if not repaired timely, could stall DNA replication, leading to genome instability. To better understand the mechanism of DNA lesion replication at the biochemical level, we have attempted to reconstitute this process in Xenopus egg extracts, the only eukaryotic in vitro system that relies solely on cellular proteins for DNA replication. By using a plasmid DNA that carries a site-specific apurinic/apyrimidinic (AP) lesion as template, we have found that DNA replication is stalled one nucleotide before the lesion. The stalling is temporary and the lesion is eventually replicated by both an error-prone mechanism and an error-free mechanism. This is the first biochemical system that recapitulates efficiently and faithfully all major aspects of DNA lesion replication. It has provided the first direct evidence for the existence of an error-free lesion replication mechanism and also demonstrated that the error-prone mechanism is a major contributor to lesion replication.


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