Global regulation of genome duplication in eukaryotes: an overview from the epifluorescence microscope

Global regulation of genome duplication in eukaryotes: an overview from the epifluorescence microscope.

John Herrick1 and Aaron Bensimon2

1. e-mail: jhenryherrick@yahoo.fr Genomic Vision 29, rue Faubourg St. Jacques 75014 Paris

2. e-mail : aaron.bensimon@genomicvision.com Genomic Vision 29, rue Faubourg St. Jacques 75014 Paris

In Chromosoma : DOI: 10.1007/s00412-007-0145-1

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Abstract

In eukaryotes, DNA replication is initiated along each chromosome at multiple sites called replication origins. Locally, each replication origin is “licensed”, or specified, at the end of the M and the beginning of G1 phases of the cell cycle. During S phase when DNA synthesis takes place, origins are activated in stages corresponding to early and late replicating domains. The staged and progressive activation of replication origins reflects the need to maintain a strict balance between the number of active replication forks and the rate at which DNA synthesis proceeds. This suggests that origin densities (frequency of initiation) and replication fork movement (rates of elongation) must be co-regulated in order to guarantee the efficient and complete duplication of each sub-chromosomal domain. Emerging evidence supports this proposal and suggests that the ATM/ATR intra-S phase checkpoint plays an important role in the co-regulation of initiation frequencies and rates of elongation. In the following, we review recent results concerning the mechanisms governing the global regulation of DNA replication and discuss the roles these mechanisms play in maintaining genome stability during both a normal and perturbed S phase.

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Introduction: Early single molecule studies of DNA replication

DNA replication has been the focus of study on extended molecules for over 40 years. DNA fibre autoradiography and electron microscopy were the principal technologies used to study the organization of DNA replication on individual molecules spread over a surface. J. Cairns first developed DNA fibre analysis in the 1960s in order to study the replication of the Escherichia coli chromosome (Cairns 1963). Later, other researchers employed electron microscopy to image “replication bubbles”, or circles of newly replicated DNA formed between un-replicated sequences (Blumenthal 1974). Both techniques provided the first quantitative assessment of replicon sizes and replication fork movement in the metazoan genome.

Huberman and Riggs later applied the method to study DNA replication in mammalian cells (Huberman and Riggs 1966; reviewed in Edenberg and Huberman 1975). These studies formed the basis of the original paradigm concerning the organization of DNA replication in the metazoan genome (reviewed in Berezney 2000). According to the model developed during these studies, the metazoan genome is organized in multiple, tandem units of replication, termed replicons. A replicon is defined as a sequence of DNA that is replicated from a single site, or origin, where DNA synthesis starts, and its size corresponds to the length of DNA replicated from the origin. Following replication initiation, DNA synthesis proceeds either bi- directionally or uni-directionally until advancing replication forks from adjacent replicons merge and replication terminates at random sites. A central tenet of the paradigm involves the

4 organization of replicons into groups, or clusters, of four to ten origins that initiate replication more or less synchronously.

The introduction of fluorescently labelled nucleotides and antibodies along with improved stretching techniques such as molecular combing has resulted in a far more efficient and reliable method for studying genome organization during DNA replication (Jackson and Pombo 1998; Herrick and Bensimon 1999). Fibre fluorography consists of using modified nucleotides such as BrdU, CldU and IdU to label actively replicating sites in the genome. The incorporated nucleotides are then detected on stretched DNA with fluorescently labelled antibodies. Following antibody detection, the labelled DNA is visualized in an epifluorescence microscope as a tandem array of discrete linear signals whose lengths can be directly measured. Initial fluorographic studies confirmed the original autoradiography findings regarding replicon sizes and clustering, and revealed that replicon clusters labelled at the beginning of one S phase were also labelled at the beginning of the following S phase (Jackson and Pombo 1998). Based on these experiments, it was concluded that replicon organization is transmitted and stably maintained from one somatic generation to the next.

Using the fluorographic approach, initial studies on embryonic genome duplication in the Xenopus laevis in vitro replication system revealed that replication origins are stochastically and asynchronously activated at int

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