**3. Two-dimensional agarose gel electrophoresis**

The movement of a DNA molecule through an agarose gel is determined either by factors intrinsic to the electrophoretic conditions (agarose concentration, the strength of the electric field, the presence of intercalating agents, etc.) as well as the size and shape of the molecule.

The most evident example of the influence of the shape of a DNA molecule on the electrophoretic mobility in an agarose gel is observed when circular DNA molecules are analyzed: supercoiled DNA molecules and the corresponding relaxed-nicked DNA forms do not migrate necessarily at the same position than a linear DNA molecule of the same mass.

Taking into account this property, neutral/neutral 2D agarose gel electroforesis technique was developed to study the shape of recombination intermediates (Bell & Byers, 1983). 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.; 2003), 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., 2006; Mirkin 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 (Viguera et al.; 2000; 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.

2D agarose gel electrophoresis consists of two successive electrophoreses in which the second dimension occurs perpendicular to the first. 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

Analysis of Chromosomal Replication Progression by Gel Electrophoresis 239

analyzed to confirm that the signal corresponding to the accumulated molecules move along

Recently, this technique was used to get insight into the nature of the elements that causes the trapping of the DNA in PFGE experiments in *E. coli* mutant strains containing an inverted *rrn* operon (Boubakri et al., 2010). No RIs were detected in the non-inverted strains or the inversion mutants that express all helicases. However, a simple-Y arc that corresponds to the accumulated Y-shape restriction intermediates was detected in all *dinG*, *rep* and *uvrD* helicase mutants in which the Inv-fragment was trapped in PFGE wells. Moreover, an intense elongated spot was observed over the simple-Y arc. These results indicate that a specific accumulation of RIs occur at the 3' end of the *rrn*

Fig. 6. Replication forks are arrested in inverted *rrn*. 2D-Gels were used to examine DNA replication in restriction fragments containing a large 3' region of *rrnA* in *InvA* mutants and of *rrnE* in *InvBE* mutants (Adapted from Boubakri et al., 2010). (A) Schematic representation of the restriction fragment used for 2D gels (only *InvBE* is presented in this figure). The position of rrn and restriction sites is shown. (B) DNA from *InvBE dinG rep* mutant was digested with *BmgBI* , analyzed by 2D gels and probed for the sequence just downstream of *rrnE*. A simple-Y arc is clearly detected. On top of this arc, an enlarged signal corresponding to arrested forks is detected. (C) A simulation of replication arrest in this fragment of about 500 pb around the *rrn* transcription terminator sequence was obtained by using the 2D-Gel computer program

the arc.

operon (Figure 6).

(Viguera et al., 1998).

branched DNA molecule like a recombination or a replication intermediate is separated from a linear molecule of the same mass during the second dimension.

As DNA replication is a continuous process, a sample of DNA isolated from an exponentially growing culture should contain all the replication intermediates (RIs), ranging from the linear non-replicative forms (named 1.0X) to molecules almost completely replicated (2.0X) (Fig. 5A). See (Krasilnikova & Mirkin, 2004), for a detailed protocol of isolation of RIs in *E. coli* and *S. cerevisiae*.

The different migration patterns of a RI digested with a specific restriction enzyme are revealed after southern blotting hybridization with a specific probe and it indicates the mode it has been replicated (Fig. 5). Electrophoresis conditions must be adapted to the fragment size in order to obtain a good separation of the different patterns (Friedman & Brewer, 1995). Different situations can be discerned by using 2D gels. (i) A single fork that moves from one end to the other end of the fragment generates a simple-Y pattern indicating that the DNA fragment is replicated passively and does not contain neither a replication origin nor a replication terminus (Fig. 5A). (ii) Two forks that move convergently generate a double-Y pattern, indicating that replication termination occurs within the analyzed fragment (Fig. 5C); and (iii) two forks that have initiated at some specific point in the analyzed fragment and progress divergently, generate a bubble pattern, indicating that DNA replication has been initiated inside this fragment (Fig. 5B).

Fig. 5. 2-D gel hybridization patterns generated by replication and recombination intermediates after two-dimensional agarose gel electrophoresis. Replication and recombination intermediates of the restriction fragment are shown above the different 2D-gel pattern. In panels B, C and D, the simple-Y arc is presented as a reference. See text for details.

Moreover, the relative proportion of a particular RI in the population increases as a consequence of the stalling of the replication forks at a specific site. This accumulation is detected as a discrete signal on top of the corresponding arc produced by the RIs. In order to map the region where replication is paused, different restriction fragments must be

branched DNA molecule like a recombination or a replication intermediate is separated

As DNA replication is a continuous process, a sample of DNA isolated from an exponentially growing culture should contain all the replication intermediates (RIs), ranging from the linear non-replicative forms (named 1.0X) to molecules almost completely replicated (2.0X) (Fig. 5A). See (Krasilnikova & Mirkin, 2004), for a detailed protocol of

The different migration patterns of a RI digested with a specific restriction enzyme are revealed after southern blotting hybridization with a specific probe and it indicates the mode it has been replicated (Fig. 5). Electrophoresis conditions must be adapted to the fragment size in order to obtain a good separation of the different patterns (Friedman & Brewer, 1995). Different situations can be discerned by using 2D gels. (i) A single fork that moves from one end to the other end of the fragment generates a simple-Y pattern indicating that the DNA fragment is replicated passively and does not contain neither a replication origin nor a replication terminus (Fig. 5A). (ii) Two forks that move convergently generate a double-Y pattern, indicating that replication termination occurs within the analyzed fragment (Fig. 5C); and (iii) two forks that have initiated at some specific point in the analyzed fragment and progress divergently, generate a bubble pattern, indicating that

from a linear molecule of the same mass during the second dimension.

DNA replication has been initiated inside this fragment (Fig. 5B).

Fig. 5. 2-D gel hybridization patterns generated by replication and recombination intermediates after two-dimensional agarose gel electrophoresis. Replication and recombination intermediates of the restriction fragment are shown above the different 2D-gel pattern. In panels B, C and D, the simple-Y arc is presented as a reference. See text

Moreover, the relative proportion of a particular RI in the population increases as a consequence of the stalling of the replication forks at a specific site. This accumulation is detected as a discrete signal on top of the corresponding arc produced by the RIs. In order to map the region where replication is paused, different restriction fragments must be

isolation of RIs in *E. coli* and *S. cerevisiae*.

for details.

analyzed to confirm that the signal corresponding to the accumulated molecules move along the arc.

Recently, this technique was used to get insight into the nature of the elements that causes the trapping of the DNA in PFGE experiments in *E. coli* mutant strains containing an inverted *rrn* operon (Boubakri et al., 2010). No RIs were detected in the non-inverted strains or the inversion mutants that express all helicases. However, a simple-Y arc that corresponds to the accumulated Y-shape restriction intermediates was detected in all *dinG*, *rep* and *uvrD* helicase mutants in which the Inv-fragment was trapped in PFGE wells. Moreover, an intense elongated spot was observed over the simple-Y arc. These results indicate that a specific accumulation of RIs occur at the 3' end of the *rrn* operon (Figure 6).

Fig. 6. Replication forks are arrested in inverted *rrn*. 2D-Gels were used to examine DNA replication in restriction fragments containing a large 3' region of *rrnA* in *InvA* mutants and of *rrnE* in *InvBE* mutants (Adapted from Boubakri et al., 2010). (A) Schematic representation of the restriction fragment used for 2D gels (only *InvBE* is presented in this figure). The position of rrn and restriction sites is shown. (B) DNA from *InvBE dinG rep* mutant was digested with *BmgBI* , analyzed by 2D gels and probed for the sequence just downstream of *rrnE*. A simple-Y arc is clearly detected. On top of this arc, an enlarged signal corresponding to arrested forks is detected. (C) A simulation of replication arrest in this fragment of about 500 pb around the *rrn* transcription terminator sequence was obtained by using the 2D-Gel computer program (Viguera et al., 1998).

Analysis of Chromosomal Replication Progression by Gel Electrophoresis 241

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