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**13** 

*Spain* 

**Analysis of Chromosomal Replication** 

*2Área de Genética, Facultad de Ciencias, Universidad de Málaga, Málaga* 

An absolute requirement for life is the preservation of genome integrity and the faithful duplication of chromosomes before segregation. A proliferating cell must duplicate its entire complement of DNA with exquisite precision facing a barrage of impediments of different nature. Single-strand breaks (SSBs) in the template DNA, either pre-existing or arising from abnormal DNA structures (folded DNA, cruciform structures or cross-links, etc.), collisions with DNA-bound proteins such as transcription complexes or DNA structural barriers restrain replication progression. For instance, during *Escherichia coli* DNA replication, the two forks initiated at the single origin of replication, *oriC*, move along the chromosome with high probability of pausing, stalling or even collapse (Maisnier-Patin et al., 2001). Replication arrest is a source of genetic instability in all types of living cells (Michel, 2000; Carr, 2002; Kolodner et al., 2002). As a consequence, cells have developed several effective strategies to tackle with replication fork arrest and/or repairing the double strand breaks (DSBs) generated at the stalled replication forks (Bierne et al., 1994; Kuzminov, 1995). Considerable evidence has been accumulated in the past decade demonstrating the involvement of recombination proteins in either direct or bypass repair of the lesions or structures blocking replication fork progression (reviewed in Courcelle et al., 2004; Kreuzer,

Conventional agarose DNA electrophoresis is one of the most frequently used techniques in molecular biology for the isolation or identification of DNA fragments. However, Pulse Field Gel Electrophoresis (PFGE) and Two-Dimensional (2D) Agarose Gel Electrophoresis techniques have been used to study biological processes such as the progression of the replication fork along a DNA fragment. In this work we introduce how these techniques has been used in bacteria to (i) verify and quantify the presence of stalled replication forks (ii) recognize DNA structure at the stalled replication fork, and (iii) understand how the

Separation of DNA fragments by standard agarose gel electrophoresis is based on the capacity of the molecules to pass through the pore generated inside the matrix gel. Using

2005; Hanawalt, 2007; Michel et al., 2007 and references herein).

replication fork could be restarted.

**2. Pulse Field Gel Electrophoresis (PFGE)** 

**1. Introduction** 

**Progression by Gel Electrophoresis** 

*1Departamento de Bioquímica, Biología Molecular y Genética Facultad de Ciencias, Universidad de Extremadura, Badajoz* 

Elena C. Guzmán1 and Enrique Viguera2

