Current Topics in Developmental Biology
Series Editor Gerald P. Schatten Director, PITTSBURGH DEVELOPMENTAL CENTER Deputy Director, Magee-Women’s Research Institute Professor and Vice-Chair of Ob-Gyn-Reproductive Sci. & Cell Biol.-Physiology University of Pittsburgh School of Medicine Pittsburgh, PA 15213
Editorial Board Peter Gru¨ss Max-Planck-Institute of Biophysical Chemistry Go¨ttingen, Germany
Philip Ingham University of Sheffield, United Kingdom
Mary Lou King University of Miami, Florida
Story C. Landis National Institutes of Health National Institute of Neurological Disorders and Stroke Bethesda, Maryland
David R. McClay Duke University, Durham, North Carolina
Yoshitaka Nagahama National Institute for Basic Biology, Okazaki, Japan
Susan Strome Indiana University, Bloomington, Indiana
Virginia Walbot Stanford University, Palo Alto, California
Founding Editors A. A. Moscona Alberto Monroy
Current Topics in Developmental Biology Volume 55 Edited by
Gerald P. Schatten Director, PITTSBURGH DEVELOPMENTAL CENTER Deputy Director, Magee-Women’s Research Institute Professor and Vice-Chair of Ob-Gyn-Reproductive Sci. & Cell Biol.-Physiology University of Pittsburgh School of Medicine Pittsburgh, PA 15213
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Cover Photo Credit: Zebrafish Nodal signaling mutant. Frontal view of Nodal signaling mutant at approximately 1 day postfertilization. See chapter 3 figure 1 WT for futher details.
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1 The Dynamics of Chromosome Replication in Yeast Isabelle A. Lucas and M. K. Raghuraman I. II. III. IV. V. VI. VII.
Introduction 1 Assays for Origin Activity 2 Organization of the Genome for Replication 11 Assembly and Activation of the Initiation Complex The Temporal Program of Origin Activation 38 Monitoring the Replication Program 49 Concluding Thoughts 59 References 59
2 Micromechanical Studies of Mitotic Chromosomes M. G. Poirier and John F. Marko I. II. III. IV. V. VI.
Introduction 76 Architecture and Components of Eukaryote Chromosomes Stretching Elasticity of Chromosomes 94 Bending Elasticity of Chromosomes 104 Viscoelasticity of Chromosomes 112 Combined Biochemical–Micromechanical Study of Mitotic Chromosomes 116 VII. Conclusion 125 References 133
3 Patterning of the Zebrafish Embyro by Nodal Signals Jennifer O. Liang and Amy L. Rubinstein I. II. III. IV. V. VI. VII.
Introduction 143 Zebrafish Nodal Signals 144 Nodal Signaling Pathway 146 Patterning the Mesoderm and Endoderm 153 Role of Nodal in Patterning the Ventral Nervous System Patterning the Left–Right Axis 162 Future Directions 165 References 165
4 Folding Chromosomes in Bacteria: Examining the Role of Csp Proteins and Other Small Nucleic Acid-Binding Proteins Nancy Trun and Danielle Johnston I. Introduction 173 II. Small Nucleic Acid-Binding Proteins Implicated in Chromosome Folding in Escherichia coli 175 III. Csp Proteins in Escherichia coli 180 IV. Relationships among Escherichia coli Csp Proteins 189 V. Csp Proteins of Bacillus Subtilis 190 VI. The Crystal Structures of CspA and Related Proteins 191 VII. Distribution of Small DNA-Binding Proteins in Archaea and Bacteria 197 VIII. Conclusions 198 References 200
Index 203 Contents of Previous Volumes
Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin.
Danielle Johnston (173), Department of Biological Sciences, Duquesne University, Pittsburgh, Pennsylvania 15219 Jennifer O. Liang (143), Biology Department, Case Western Reserve University, Cleveland, Ohio 44106 Isabelle A. Lucas (1), Department of Genome Sciences, University of Washington, Seattle, Washington 98195 John F. Marko (75), Department of Physics and Bioengineering, University of Illinois at Chicago, Chicago, Illinois 60607 M. G. Poirier (75), Department of Physics, University of Illinois and Chicago, Chicago, Illinois 60607 Mokur K. Raghuraman (1), Department of Genome Sciences, University of Washington, Seattle, Washington 98195 Amy L. Rubinstein (143), Zygogen, Atlanta, Georgia 30303 Nancy Trun (173), Department of Biological University, Pittsburgh, Pennsylvania 15219
The Dynamics of Chromosome Replication in Yeast Isabelle A. Lucas and M. K. Raghuraman Department of Genome Sciences University of Washington Seattle, Washington 98195
I. Introduction II. Assays for Origin Activity A. The Autonomously Replicating Sequence Assay B. Two-Dimensional Agarose Gel Electrophoresis C. Alkaline Gel Electrophoresis Assay for Nascent DNA Strands D. Density Transfer Method to Determine the Time of Replication E. Single Molecule Analysis by Molecular Combing F. Assaying Replication on a Genomic Scale G. Strengths and Limitations of the Assays III. Organization of the Genome for Replication A. Origin Structure B. Origin Spacing and Location IV. Assembly and Activation of the Initiation Complex A. Formation of the Prereplicative Complex B. Origin Firing C. Preventing Reinitiation D. What Determines Origin Choice and EYciency? E. Fork Migration and Termination V. The Temporal Program of Origin Activation A. Setting Up and Reading the Temporal Program B. What Is the Physiological Relevance of the Temporal Program? VI. Monitoring the Replication Program A. The S Phase Response to DNA Damage and Hydroxyurea B. Detecting Ongoing, Uninterrupted Replication VII. Concluding Thoughts References
I. Introduction Chromosomal DNA replication is a task of immense complexity. No sooner has a cell divided than it begins preparation for the next round of DNA replication. Origins of replication, sites that will direct initiation of DNA synthesis at roughly 30- to 100-kb intervals along the chromosome, Current Topics in Developmental Biology, Vol. 55 Copyright 2003, Elsevier (USA). All rights reserved. 0070-2153/03 $35.00
Lucas and Raghuraman
begin to recruit the proteins that will be required for the initial steps in DNA synthesis. By the time the cells reach START, a critical decision point late in G1 phase of the cell cycle, origins will have completed the first phase of this assembly process and become licensed for initiation of DNA synthesis. DNA synthesis begins in S phase with the unwinding of DNA by replication initiation complexes and the assembly of replication forks. As replication proceeds, the forks traverse the genome, unraveling the structure of the chromosomes as they advance and leaving repackaged and duplicated DNA in their wake. Every base pair must be faithfully copied, yet no portion of the genome may be copied more than once per cell cycle. This task is made more diYcult by the temporal program of origin activation that eukaryotes follow. Origins are activated in a reproducible, sequential pattern through S phase and then must be shut oV for the remainder of the S phase even as other origins become active. How cells solve this problem of preventing reinitiation at origins that have already initiated has become clear in the last few years, but how the temporal program is laid down in the first place remains unanswered. Even more obscure is the question of why cells bother with this temporal program at all. Work on the budding yeast Saccharomyces cerevisiae has led the way in our exploration of how replication is choreographed. As with many other aspects of biology, the basic framework underlying the process of replication is conserved between yeast and higher eukaryotes, including humans. From the recent explosion of work on yeast and other model systems, we have begun to have a deeper appreciation of the dynamic aspects of replication—how the control of replication initiation is tied to phases of the cell cycle, how the activation of diVerent origins within the genome are coordinated, and how checkpoint surveillance systems monitor and ensure the orderly progress and completion of S phase. This chapter reviews our current understanding of how the program of replication is laid down and carried out in S. cerevisiae, with particular emphasis on the dynamic aspects of replication and the open questions still remaining.
II. Assays for Origin Activity With any endeavor in science, the sophistication of the questions that can be asked is limited by the techniques available, and replication is no exception. The interpretation of experimental results also hinges on a clear understanding of the methods used and their limitations. We shall begin, therefore, with a brief overview of the methods that are used most commonly in studying replication.
1. The Dynamics of Chromosome Replication in Yeast
A. The Autonomously Replicating Sequence Assay Replication origins had been observed previously in yeast by methods such as electron microscopy and fiber autoradiography (e.g., [1,2]), but identification of specific sequences that can function as replication origins first came from a genetic assay that required stable maintenance of a plasmid [3–5]. In this assay, DNA fragments are tested for their ability to confer autonomous replication on a plasmid that otherwise cannot be maintained as an extrachromosomal element in yeast. Sequences that support maintenance of the plasmid presumably do so by acting as origins of replication. Potential origins of replication can thus be identified in a very simple transformation assay. The autonomously replicating sequences (ARS elements) so identified are small, on the order of 100–200 bp . They have been shown to function as origins of replication on plasmids in vivo (see later; [7,8]) and to various extents as origins on chromosomes. For example, ARS301 is capable of supporting maintenance of a plasmid but is considered to be a ‘‘silent’’ origin in its native context on chromosome III, i.e., it shows no detectable origin activity in the chromosome [6,9,10]. Thus, the ARS assay allows identification of potential origins of replication; other means (see later) must be used to test for origin activity on the chromosome.
B. Two-Dimensional Agarose Gel Electrophoresis Developed in 1987, the two-dimensional (2-D) agarose gel electrophoresis technique has dramatically changed how we look at replication . Distinct patterns of gel migration are observed in this technique for diVerent forms of replication intermediates (Fig. 1a). These patterns allow recognition of a restriction fragment that contains an active origin (bubble-shaped replication intermediates), a fork passing through (Y-shaped intermediates), or two forks converging (double Y intermediates) (Fig. 1a). The eYciency of an origin is defined as the percentage of cell cycles in which it initiates replication or ‘‘fires.’’ The classical 2-D gel method described earlier only permits qualitative estimates of origin eYciency. A modification of this technique, fork direction analysis [11,12], allows the quantitation of origin eYciency by revealing the proportion of leftward versus rightward moving forks in a restriction fragment. By assaying fork movement on either side of an origin, one can measure the fraction of cells in which forks are moving outward from the origin (initiation event) compared to those in which forks enter the region (no initiation). Using this method, origin firing can be ranked from highly eYcient (e.g., ARS607 fires in 85% of cells) to ineYcient (e.g., ARS605 fires in 2N DNA content.
Lucas and Raghuraman
However, this phenotype was seen only when the ORC mutations were combined with disruptions in the normal regulation of Mcm and Cdc6p (Mcm kept in the nucleus by fusion of a nuclear localization signal to Mcm7p, and Cdc6p blocked from degradation using a partially stabilized allele) . No pairwise combination of disruptions was suYcient to cause overreplication, and even the triply disrupted strain did not undergo a complete extra round of replication; 2-D gel analysis detected reinitiation at some origins but not at others . These results highlight the elaborately redundant mechanisms that cells have evolved to prevent rereplication. More recently, yet another mechanism has been uncovered—one involving the depletion of Cdt1p from the nucleus. As described earlier, Cdt1p is needed along with Cdc6p to recruit Mcms to the ORC complex at origins [101,102]. Like Mcms, Cdt1p is also exported from the nucleus during S phase, and this export is blocked by inhibition of Clb-CDK by the ectopic expression of Sic1p . This newly discovered mode of CDK-dependent control may explain the failure to get a complete extra round of replication in the experiments described earlier . Preventing reinitiation must be a particularly high priority in higher eukaryotes, which have evolved a completely separate, CDK-independent mechanism to regulate Cdt1p activity—one mediated by geminin, a specific inhibitor of Cdt1p that is destroyed during G1 to allow activation of Cdt1p .
D. What Determines Origin Choice and Efficiency? Origin eYciency on the chromosome can be high or low—some origins function in almost every cell cycle, whereas others are used less frequently. For example, of the nine ARSs mapped on chromosome VI [13,174], only four are used in at least 50% of the cell cycles; the others are used variously at eYciencies of 10–40% . Likewise, 11 of the 19 potential origins on chromosome III are used in