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J. Biol. Chem., Vol. 283, Issue 19, 13341-13356, May 9, 2008

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Single-stranded DNA-binding Protein in Vitro Eliminates the Orientation-dependent Impediment to Polymerase Passage on CAG/CTG Repeats^*

Emmanuelle Delagoutte¹, Geoffrey M. Goellner, Jie Guo, Giuseppe Baldacci², and Cynthia T. McMurray²³

From the Génotoxicologie et Cycle Cellulaire, Institut Curie, CNRS, Université Paris-Sud 11, 91405 Orsay Cedex, France, and the Departments of Molecular Pharmacology and Experimental Therapeutics, and Biochemistry and Molecular Biology, Mayo Medical Foundation, Rochester, Minnesota 55905

Small insertions and deletions of trinucleotide repeats (TNRs) can occur by polymerase slippage and hairpin formation on either template or newly synthesized strands during replication. Although not predicted by a slippage model, deletions occur preferentially when 5'-CTG is in the lagging strand template and are highly favored over insertion events in rapidly replicating cells. The mechanism for the deletion bias and the orientation dependence of TNR instability is poorly understood. We report here that there is an orientation-dependent impediment to polymerase progression on 5'-CAG and 5'-CTG repeats that can be relieved by the binding of single-stranded DNA-binding protein. The block depends on the primary sequence of the TNR but does not correlate with the thermodynamic stability of hairpins. The orientation-dependent block of polymerase passage is the strongest when 5'-CAG is the template. We propose a "template-push" model in which the slow speed of DNA polymerase across the 5'-CAG leading strand template creates a threat to helicase-polymerase coupling. To prevent uncoupling, the TNR template is pushed out and by-passed. Hairpins do not cause the block, but appear to occur as a consequence of polymerase pass-over.

Received for publication, January 7, 2008 , and in revised form, February 5, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants GM 066359 and NS40738 (to C. T. M.). This work was also supported by the Mayo Foundation, CNRS, and Institut Curie (Réplication, Instabilité Chromosomique et Cancer; to G. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

² These authors contributed equally to this work.

¹ To whom correspondence may be addressed. Tel.: 33-1-69-86-30-34; Fax: 33-1-69-86-94-29; E-mail: emmanuelle.delagoutte{at}curie.u-psud.fr.

³ To whom correspondence may be addressed: Mayo Clinic, 200 First St., SW Guggenheim 7, Rochester, MN 55905. Tel.: 507-284-1597; Fax: 507-284-9111; E-mail: mcmurray.cynthia{at}mayo.edu.