Table of Contents
Molecular Biology International
Volume 2011 (2011), Article ID 213824, 14 pages
http://dx.doi.org/10.4061/2011/213824
Research Article

5CAG and 5CTG Repeats Create Differential Impediment to the Progression of a Minimal Reconstituted T4 Replisome Depending on the Concentration of dNTPs

1Muséum National d'Histoire Naturelle, Département “Régulations, Développement et Diversité Moléculaire”, Laboratoire de Régulations et Dynamique des Génomes, USM 0503—INSERM U 565—UMR 7196, Case Postale no 26, 57 rue Cuvier, 75231 Paris cedex 05, France
2Institut Jacques Monod, UMR7592, “Pathologies de la réplication de l'ADN”, CNRS and Université Paris-Diderot, 15 Rue Hélène Brion, 75205 Paris Cedex 13,, France

Received 3 February 2011; Accepted 13 May 2011

Academic Editor: Giuseppina Giglia-Mari

Copyright © 2011 Emmanuelle Delagoutte and Giuseppe Baldacci. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Instability of repetitive sequences originates from strand misalignment during repair or replicative DNA synthesis. To investigate the activity of reconstituted T4 replisomes across trinucleotide repeats (TNRs) during leading strand DNA synthesis, we developed a method to build replication miniforks containing a TNR unit of defined sequence and length. Each minifork consists of three strands, primer, leading strand template, and lagging strand template with a 5 single-stranded (ss) tail. Each strand is prepared independently, and the minifork is assembled by hybridization of the three strands. Using these miniforks and a minimal reconstituted T4 replisome, we show that during leading strand DNA synthesis, the dNTP concentration dictates which strand of the structure-forming 5CAG/5CTG repeat creates the strongest impediment to the minimal replication complex. We discuss this result in the light of the known fluctuation of dNTP concentration during the cell cycle and cell growth and the known concentration balance among individual dNTPs.