John Herrick

Kinetic model of DNA replication in eukaryotic organisms

We formulate a kinetic model of DNA replication that quantitatively describes recent results on DNA replication in the in vitro system of Xenopus laevis prior to the mid-blastula transition. The model describes well a large amount of different data within a simple theoretical framework. This allows one, for the first time, to determine the parameters governing the DNA replication program in a eukaryote on a genome-wide basis. In particular, we have determined the frequency of origin activation in time and space during the cell cycle. Although we focus on a specific stage of development, this model can easily be adapted to describe replication in many other organisms, including budding yeast.

Single molecule analysis of DNA replication

We describe here a novel approach for the study of DNA replication. The approach is based on a process called molecular combing and allows for the genome wide analysis of the spatial and temporal organization of replication units and replication origins in a sample of genomic DNA. Molecular combing is a process whereby molecules of DNA are stretched and aligned on a glass surface by the force exerted by a receding air/water interface. Since the stretching occurs in the immediate vicinity of the meniscus, all molecules are identically stretched in a size and sequence independent manner. The application of fluorescence hybridization to combed DNA results in a high resolution (1 to 4 kb) optical mapping that is simple, controlled and reproducible. The ability to comb up to several hundred haploid genomes on a single coverslip allows for a statistically significant number of measurements to be made. Direct labeling of replicating DNA sequences in turn enables origins of DNA replication to be visualized and mapped. These features therefore make molecular combing an attractive tool for genomic studies of DNA replication. In the following, we discuss the application of molecular combing to the study of DNA replication and genome stability.

Persistence Length of Chromatin Determines Origin Spacing in Xenopus Early-Embryo DNA Replication: Quantitative Comparisons between Theory and Experiment

In Xenopus early embryos, replication origins neither require specific DNA sequences nor is there an efficient S/M checkpoint, even though the whole genome (3 billion bases) is completely duplicated within 10-20 minutes. This leads to the “random-completion problem” of DNA replication in embryos, where one needs to find a mechanism that ensures complete, faithful, timely reproduction of the genome without any sequence dependence of replication origins. We analyze recent DNA replication data in Xenopus laevis egg extracts and find discrepancies with models where replication origins are distributed independently of chromatin structure. Motivated by these discrepancies, we have investigated the role that chromatin looping may play in DNA replication. We find that the loop-size distribution predicted from a wormlike-chain model of chromatin can account for the spatial distribution of replication origins in this system quantitatively. Together with earlier findings of increasing frequency of origin firings, our results can explain the random-completion problem. The agreement between experimental data (molecular combing) and theoretical predictions suggests that the intrinsic stiffness of chromatin loops plays a fundamental biological role in DNA replication in early-embryo Xenopus in regulating the origin spacing.

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

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 the G1 phases of the cell cycle. During the 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 coregulated to guarantee the efficient and complete duplication of each subchromosomal domain. Emerging evidence supports this proposal and suggests that the ATM/ATR intra-S phase checkpoint plays an important role in the coregulation of initiation frequencies and rates of elongation. In this paper, 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.

Invited Review. Imaging of Single DNA Molecule: Applications to High-Resolution Genomic Studies

Single molecule analysis of DNA has revealed new insights into its structural and physical properties. The application of new methods for manipulating and visualizing DNA has resulted in important advances in high-resolution physical mapping of the genome and quantitative cytogenetic studies of genomic abnormalities (Lichter 1997). Studies of single molecules of DNA have employed a variety of approaches including electron microscopy, atomic force microscopy, scanning-tunneling microscopy and fluorescence microscopy. A number of new technologies have recently been developed to exploit fluorescence microscopys full potential for genomic analysis and the fine mapping of subtle genetic alterations. In the case of the latter application, particular emphasis has been placed on developing new methods for stretching DNA for high-resolution fluorescence in-situ hybridization studies. We have recently described a process called molecular combing according to which single DNA molecules bound by their extremities to a solid surface are uniformly stretched and aligned by a receding air/water interface (Bensimon et al. 1994). In the following, we will review recent developments concerning molecular combing and discuss its current and potential applications for the high-resolution mapping of the human genome, the detection and quantification of subtle genomic imbalances and the positional cloning of disease-related genes.

Introduction to Molecular Combing: Genomics, DNA Replication, and Cancer

The sequencing of the human genome inaugurated a new era in both fundamental and applied genetics. At the same time, the emergence of new technologies for probing the genome has transformed the field of pharmaco-genetics and made personalized genomic profiling and high-throughput screening of new therapeutic agents all but a matter of routine. One of these technologies, molecular combing, has served to bridge the technical gap between the examination of gross chromosomal abnormalities and sequence-specific alterations. Molecular combing provides a new perspective on the structure and dynamics of the human genome at the whole genome and sub-chromosomal levels with a resolution ranging from a few kilobases up to a megabase and more. Originally developed to study genetic rearrangements and to map genes for positional cloning, recent advances have extended the spectrum of its applications to studying the real-time dynamics of the replication of the genome. Understanding how the genome is replicated is essential for elucidating the mechanisms that both maintain genome integrity and result in the instabilities leading to human genetic disease and cancer. In the following, we will examine recent discoveries and advances due to the application of molecular combing to new areas of research in the fields of molecular cytogenetics and cancer genomics.

Unscheduled DNA replication origin activation at inserted HPV 18 sequences in a HPV-18/MYC amplicon.

Oncogene amplification is a critical step leading to tumorigenesis, but the underlying mechanisms are still poorly understood. Despite data suggesting that DNA replication is a major source of genomic instability, little is known about replication origin usage and replication fork progression in rearranged regions. Using a single DNA molecule approach, we provide here the first study of replication kinetics on a previously characterized MYC/papillomavirus (HPV18) amplicon in a cervical cancer. Using this amplicon as a model, we investigated the role DNA replication control plays in generating amplifications in human cancers. The data reveal severely perturbed DNA replication kinetics in the amplified region when compared with other regions of the same genome. It was found that DNA replication is initiated from both genomic and viral sequences, resulting in a higher median frequency of origin firings. In addition, it was found that the higher initiation frequency was associated with an equivalent increase in the number of stalled replication forks. These observations raise the intriguing possibility that unscheduled replication origin activation at inserted HPV‐18 viral DNA sequences triggers DNA amplification in this cancer cell line and the subsequent overexpression of the MYC oncogene. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045‐2257/suppmat.