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Evaluating genome-scale approaches to eukaryotic DNA replication

Key Points

  • Mechanisms regulating where and when eukaryotic DNA replication initiates remain a fundamental mystery in molecular biology.

  • Genome-scale approaches are now being used to identify the location of replication origins and to evaluate replication timing.

  • Mapping replication origins in yeasts has been successful, but the current data sets from multicellular organisms are scarce and inconsistent.

  • Methods for mapping origins include trapping the earliest replicated DNA by replication fork arrest, mapping small nascent leading strands and trapping replication bubbles.

  • Studies of replication timing can involve prospective or retroactive synchronization of cells, followed by comparison of early- and late-S-phase DNA by microarray or sequencing.

  • Genome-wide studies of replication timing have shown that timing is regulated at the level of replication domains and that there are links between replication timing and chromatin structure.

Abstract

Mechanisms regulating where and when eukaryotic DNA replication initiates remain a mystery. Recently, genome-scale methods have been brought to bear on this problem. The identification of replication origins and their associated proteins in yeasts is a well-integrated investigative tool, but corresponding data sets from multicellular organisms are scarce. By contrast, standardized protocols for evaluating replication timing have generated informative data sets for most eukaryotic systems. Here, I summarize the genome-scale methods that are most frequently used to analyse replication in eukaryotes, the kinds of questions each method can address and the technical hurdles that must be overcome to gain a complete understanding of the nature of eukaryotic replication origins.

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Figure 1: How to find an origin.
Figure 2: Different methods may enrich for different origins.
Figure 3: Replication timing analysis by retroactive fluorescence-activated cell sorter synchronization.

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Acknowledgements

I thank J. Huberman, D. Macalpine, O. Aparicio, A. Beilinsky, N. Rhind, M.-N. Prioleau, A. Dutta, H. Masukata, A. Schepers, J. Hamlin, K. Plath, B. Papp, L. Mesner, M. Mechali, K. Ekwall and members of my laboratory for helpful discussions during the preparation of this article.

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FURTHER INFORMATION

A complete protocol for genome–scale analysis of replication timing in mammalian cells

DNAReplication (a knowledge base for the eukaryotic DNA replication community that includes a forum and a list of new papers)

The ENCODE Project

OriDB (a catalogue of confirmed and predicted DNA replication origin sites)

ReplicationDomain (an interactive database of replication timing and related chromosomal properties, supplemented with protocols)

Glossary

Pre-replication complex

(pre-RC). A complex of proteins that forms at the origin of replication during the initiation step of DNA replication. All pre-RC proteins are essential for DNA replication. The pre-RC is typically thought to consist of origin recognition complex (ORC), DNA replication factor Cdt1, cell division cycle protein 6 (Cdc6) and mini-chromosome maintenance (MCM) complex.

Dormant origin

Cells have a large excess of replication origins over what they need to complete DNA replication. Origins that are in the vicinity of a recently initiated origin normally will be replicated passively when the replication fork passes through them. However, if DNA damage or low-nucleotide pools slow the replication forks, these origins can fire to complete duplication of nearby DNA in a timely fashion.

Replication origin

A site where replication is initiated during S phase. It is bound by the origin recognition complex.

Replication timing programme

All eukaryotic cells replicate segments of their genomes in a defined temporal sequence. This process is referred to as replication timing. The temporal order in which segments of DNA are replicated is specific to specific cell types, and that temporal order is its replication timing programme.

Origin recognition complex

A complex of six subunits that binds to the origins of DNA replication in an ATP-dependent manner before initiation to recruit additional protein members of the pre-replication complex.

Chromatin immunoprecipitation

A technique that is used to identify the location of DNA-binding proteins and epigenetic marks in the genome. Genomic sequences containing the mark of interest are enriched by binding soluble DNA chromatin extracts (complexes of DNA and protein) to an antibody that recognizes the mark.

Mini-chromosome maintenance complex

An oligomeric complex that is suggested to be the helicase involved in replication.

Phased nucleosomes

Nucleosomes that are evenly spaced. This usually occurs when a nucleosome is positioned by a DNA sequence or chromatin protein, which restricts the possible locations of its nearest neighbours.

Efficiency

The percentage of replication cycles in which any given origin is used as an initiation site.

Replication fork

The branch-point structure that forms at the site of active DNA synthesis, where helicases break the hydrogen bonds tethering the two DNA strands and unwind the DNA.

Primer extension

Any configuration in which a partially single-stranded nucleic acid is annealed with a 5′ overhang to a smaller complementary strand. The 3′ hydroxyl of the annealed complementary strand can serve as a primer that can be extended by DNA polymerase along the remaining single-stranded portion of the larger template molecule.

DNase I hypersensitive site

A region of the genome that is readily degraded by the enzyme DNase I owing to decreased nucleosome occupancy (an 'open' chromatin structure).

Replication bubble

The structure formed where two replication forks, derived from the same replication origins, are moving bidirectionally away from the site of initiation. The intervening DNA consists of two newly synthesized strands.

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Gilbert, D. Evaluating genome-scale approaches to eukaryotic DNA replication. Nat Rev Genet 11, 673–684 (2010). https://doi.org/10.1038/nrg2830

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