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Methods in DNA replication
Methods 57 (2012) 139
Contents lists available at SciVerse ScienceDirect
Methods journal homepage: www.elsevier.com/locate/ymeth
Guest Editor’s Introduction
Methods in DNA replication
DNA replication is how our genome is reproduced at each cell division to ensure that all the information required for life is transmitted to the daughter cells. The quest for understanding its mechanism started at the biochemical level. The pioneer work on enzymology of bacterial DNA replication by Arthur Kornberg, who discovered the first DNA polymerase, was followed with the identification of the many factors involved in DNA synthesis. This biochemical analysis was the foundation of most of the methods used to characterize enzymatic reactions linked to DNA replication. It also led to fundamental applications such as DNA amplification and DNA sequencing that have revolutionized genome analysis and genomics. The concept of a replication origin also emerged from genetic experiments leading to the replicon model by Jacob, Brenner and Cuzin. Analysis of DNA replication in metazoan cells proved to be more difficult. Replication of a human genome requires 30 000–50 000 replication origins to be activated at each cell cycle, and their nature remained mysterious until very recent years. Replication of short viral genomes, such as the SV40 and Polyomavirus genomes, and then of short plasmids in yeast, or the use of in vitro systems based on the Xenopus model, paved the way to much progress in this field at the DNA or protein level. The timing of activation of DNA replication origins is now well characterized thanks to the arrival of genome-wide approaches based on micro-arrays analyses or high throughput sequencing. Similar approaches are now shedding some light to the characterization of metazoan DNA replication origins. The main eukaryotic factors involved in DNA replication are now identified, and proteomics as well as new sensitive DNA imaging and biophysical methods are now leading to the description of the complete repertoire of DNA replication features and components. The biochemical analysis of DNA replication, so well exploited for the unraveling of bacterial DNA replication, is also gaining a revival of interests with new attempts of reconstitution experiments. In this book, leading experts in this field propose thirteen chapters that describe updated molecular and biochemical methods to investigate the actors and mechanisms of DNA replication. Chapter 1 describes how the organization of DNA synthesis in cell nuclei during the S phase can be imaged.
1046-2023/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ymeth.2012.08.007
Chapter 2 describes DNA combing, a powerful method that allows the identification of DNA replication origins and the replication fork speed on stretched individual DNA molecules. Chapter 3 provides a comprehensive and precise genome-wide method to map DNA replication origins in eukaryotic cells. Chapter 4 focuses on the problems encountered when performing genome-wide analyses of replication timing. Chapter 5 describes the 2D agarose gel electrophoresis method used to detect replication origins and DNA replication intermediates at a given locus. Chapter 6 shows how chromatin immunoprecipitation (ChIP) can identify DNA binding sites of DNA replication proteins. Chapter 7 concerns a powerful in vitro system derived from Xenopus eggs to characterize new DNA replication factors and the regulation of DNA replication. Chapter 8 explains a method for the detection of replication proteins on single DNA molecules attached to a microfluidic flow cell. Chapter 9 is on the SILAC method for quantitative proteomic characterization of DNA replication proteins in yeast. Chapter 10 describes the large scale purification of replication factors through the use of insect and human cells. Chapter 11 discusses a useful method for in vitro reconstitution of pre-replication complexes from purified components. Chapter 12 explains a technique for generating conditionallethal replication mutants in yeast. Chapter 13 is a description of a high-throughput screen to identify molecules that induce re-replication or endo-replication and genes that control such mechanisms. Marcel Méchali Institute of Human Genetics, CNRS, Replication and Genome Dynamics, 141, Rue de la Cardonille, 34396 Montpellier Cedex 5, France
Contents lists available at SciVerse ScienceDirect
Methods journal homepage: www.elsevier.com/locate/ymeth
Guest Editor’s Introduction
Methods in DNA replication
DNA replication is how our genome is reproduced at each cell division to ensure that all the information required for life is transmitted to the daughter cells. The quest for understanding its mechanism started at the biochemical level. The pioneer work on enzymology of bacterial DNA replication by Arthur Kornberg, who discovered the first DNA polymerase, was followed with the identification of the many factors involved in DNA synthesis. This biochemical analysis was the foundation of most of the methods used to characterize enzymatic reactions linked to DNA replication. It also led to fundamental applications such as DNA amplification and DNA sequencing that have revolutionized genome analysis and genomics. The concept of a replication origin also emerged from genetic experiments leading to the replicon model by Jacob, Brenner and Cuzin. Analysis of DNA replication in metazoan cells proved to be more difficult. Replication of a human genome requires 30 000–50 000 replication origins to be activated at each cell cycle, and their nature remained mysterious until very recent years. Replication of short viral genomes, such as the SV40 and Polyomavirus genomes, and then of short plasmids in yeast, or the use of in vitro systems based on the Xenopus model, paved the way to much progress in this field at the DNA or protein level. The timing of activation of DNA replication origins is now well characterized thanks to the arrival of genome-wide approaches based on micro-arrays analyses or high throughput sequencing. Similar approaches are now shedding some light to the characterization of metazoan DNA replication origins. The main eukaryotic factors involved in DNA replication are now identified, and proteomics as well as new sensitive DNA imaging and biophysical methods are now leading to the description of the complete repertoire of DNA replication features and components. The biochemical analysis of DNA replication, so well exploited for the unraveling of bacterial DNA replication, is also gaining a revival of interests with new attempts of reconstitution experiments. In this book, leading experts in this field propose thirteen chapters that describe updated molecular and biochemical methods to investigate the actors and mechanisms of DNA replication. Chapter 1 describes how the organization of DNA synthesis in cell nuclei during the S phase can be imaged.
1046-2023/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ymeth.2012.08.007
Chapter 2 describes DNA combing, a powerful method that allows the identification of DNA replication origins and the replication fork speed on stretched individual DNA molecules. Chapter 3 provides a comprehensive and precise genome-wide method to map DNA replication origins in eukaryotic cells. Chapter 4 focuses on the problems encountered when performing genome-wide analyses of replication timing. Chapter 5 describes the 2D agarose gel electrophoresis method used to detect replication origins and DNA replication intermediates at a given locus. Chapter 6 shows how chromatin immunoprecipitation (ChIP) can identify DNA binding sites of DNA replication proteins. Chapter 7 concerns a powerful in vitro system derived from Xenopus eggs to characterize new DNA replication factors and the regulation of DNA replication. Chapter 8 explains a method for the detection of replication proteins on single DNA molecules attached to a microfluidic flow cell. Chapter 9 is on the SILAC method for quantitative proteomic characterization of DNA replication proteins in yeast. Chapter 10 describes the large scale purification of replication factors through the use of insect and human cells. Chapter 11 discusses a useful method for in vitro reconstitution of pre-replication complexes from purified components. Chapter 12 explains a technique for generating conditionallethal replication mutants in yeast. Chapter 13 is a description of a high-throughput screen to identify molecules that induce re-replication or endo-replication and genes that control such mechanisms. Marcel Méchali Institute of Human Genetics, CNRS, Replication and Genome Dynamics, 141, Rue de la Cardonille, 34396 Montpellier Cedex 5, France