[46] Analysis of replication intermediates by two-dimensional agarose gel electrophoresis

Checkpoint-mediated control of replisome–fork association and signalling in response to replication pausing

“…We failed to detect any abnormal replication intermediates in HU-treated rfa1-t11 cells using the twodimensional gel technique (Friedman and Brewer, 1995) (Figure 3a), suggesting that, in this mutant background, replication forks remain stable following HU treatment. This is also consistent with the observation that rfa1-t11 cells are still able to complete replication during recovery from HU (data not shown) and that the level of Orc2 and Pol a fork association is comparable to the one of wt cells (Figure 3b).…”

The N-terminal 45-kDa Domain of Dna2 Endonuclease/Helicase Targets the Enzyme to Secondary Structure DNA

“…After resuspension, the combined DNA fractions were precipitated with ethanol and sodium acetate and dissolved in 10 mM Tris-HCl (pH 8). The DNA (10 -20 g) was digested with HindIII/EcoRV or XhoI for ARS305 or ARS216, respectively, and subjected to neutral-neutral two-dimensional gel electrophoresis as described previously (37). After electrophoresis, the gels were subjected to depurination, denaturation, and neutralization as described previously (38).…”

Analysis of Chromosomal Replication Progression by Gel Electrophoresis

“…Two different migration conditions are used so that the first dimension conditions (low voltage, low agarose concentration) minimize the effect of molecular shape on electrophoretic mobility, whereas this effect is maximized during the second dimension (high voltage and high agarose concentration, in the presence of an intercalating agent) (Friedman & Brewer, 1995). As a consequence, a branched DNA molecule like a recombination or a replication intermediate is separated from a linear molecule of the same mass during the second dimension.…”

“…Later on, it was adapted to study the DNA replication intermediates (RIs) (Brewer & Fangman, 1987). Since then, 2D agarose gel electrophoresis was used to map and characterize replication origins (Brewer & Fangman, 1988;Gahn & Schildkraut, 1989;Liu & Botchan, 1990;Schvartzman et al, 1990;Linskens & Huberman, 1990 b;Friedman & Brewer, 1995;Bach et al;, to analyze the progression of DNA replication along a DNA fragment (Azvolinsky et al, 2006), to characterize replication fork barriers (Brewer & Fangman, 1988;Linskens & Huberman, 1988;Hernandez et al, 1993;Wiesendanger et al, 1994;Samadashwily et al, 1997, López-Estraño et al, 1998, Possoz et al, 2006Mirkin et al, 2006, Boubakri et al, 2010, replication termination (Zhu et al, 1992;Santamaría et al, 2000a,b), origin replication interference (Viguera et al, 1996), RIs knotting (Viguera et al, 1996;Sogo et al, 1999), fork reversal Fierro-Fernandez et al, 2007a) or the topology of partially replicated plasmids (Martín-Parras et al, 1998;Lucas et al, 2001). See (Schvartzman et al, 2010) for an excellent review in plasmid DNA replication analyzed by 2D-gel.…”