Use of DNA Combing for Studying DNA Replication In Vivo in Yeast and Mammalian Cells

Abstract

Plasticity is an inherent feature of chromosomal DNA replication in eukaryotes. Potential origins of DNA replication are made in excess, but are used (fired) in a partly stochastic, partly programmed manner throughout the S phase of the cell cycle. Since most origins have a firing efficiency below 50%, population-based analysis methods yield a cumulative picture of origin activity (obtained by accretion) that does not accurately describe how chromosomes are replicated in single cells. DNA combing is a method that allows the alignment on silanized glass coverslips, at high density and with uniform stretching, of single DNA molecules in the Mb range. If this DNA is isolated from cells that have been labelled with halogenated nucleotides (BrdU, CldU, IdU), it is possible to determine the density and position of replication origins as well as the rate and symmetry of fork progression, quantitatively and on single DNA molecules. This chapter will successively describe (a) the preparation ofsilanized coverslips, (b) the incorporation of halogenated nucleotides in newly synthesized DNA in yeast and mammalian cell lines, (c) the preparation and combing of genomic DNA, and finally (d) the acquisition and analysis of single-molecule images to extract salient features of replication dynamics.

Analysis of Replicating Yeast Chromosomes by DNA Combing

“…Steps 1-15 featured an adaptation of the liquid-phase silanization procedure described previously by Labit et al (2008). Alternative procedures for silanization in the vapor phase can also be considered (Schwob et al 2009). Note too that suitable surfaces are also available commercially (http://www .genomicvision.com).…”

“…This procedure is suitable for measuring replication fork rates and replication origin usage. The protocol (Steps 23-66) is adapted from the procedures of the Schwob and Pasero laboratories (Lengronne et al 2001;Versini et al 2003;Schwob et al 2009;Bianco et al 2012). It is also amenable to more complicated double-labeling procedures involving the sequential addition of IdU and CldU to measure replication fork stalling and fork asymmetry.…”

The Involvement of Acidic Nucleoplasmic DNA-binding Protein (And-1) in the Regulation of Prereplicative Complex (pre-RC) Assembly in Human Cells

“…Cells were then collected by trypsinization, and DNA was gently extracted for molecular combing as described previously (34). DNA was combed on silanized surfaces (Microsurfaces, Inc.) and denatured in 1 N NaOH and probed with the following primary antibodies: mouse anti-BrdU (IdU-specific; clone BD44, Becton Dickinson), rat anti-BrdU (CldU-specific; clone BU-75, AbD Serotec) and mouse anti-ssDNA (clone [16][17][18][19]Millipore).…”

“…Depletion of And-1 Reduces Both the Velocity and Density of Replication Forks-To further characterize the role of And-1 in DNA replication, we examined the progression of replication forks (fork velocity) and the number of forks per length of DNA (fork density) in siGl2-and siAnd-1-treated cells using molecular combing (34,40,41). Cells transfected with either siGl2 or siAnd-1 were labeled with 100 M IdU for 20 min and then 100 M CldU for 20 min before being harvested at 36 h after transfection.…”

Unbiased segregation of yeast chromatids in Saccharomyces cerevisiae

“…The experiments took advantage of improved strains for exogenous thymidine labeling where the cells utilize thymidine with greater efficiency (Lengronne et al 2001;Viggiani and Aparicio 2006). The cells do not delay in S phase as did previous strains, and they have been used for a variety of experiments (Viggiani et al 2010;Schwob et al 2009). The experimental design for testing biased chromosome segregation is shown in Fig.…”