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Intracellular checkpoint controls constitute a network of signal transd- tion pathways that protect cells from external stresses and internal errors. Ext- nal stresses can be generated by the continuous assault of DNA-damaging agents, such as environmental mutagens, ultraviolet (UV) light, ionizing radiation, or the reactive oxygen species that can arise during normal cellular metabolism. In response to any of these assaults on the integrity of the genome, the activation of the network of checkpoint control pathways can lead to diverse cellular responses, such as cell cycle arrest, DNA repair, or elimination of the cell by cell death (apoptosis) if the damage cannot be repaired. Moreover, internal errors can occur during the highly orchestrated replication of the cellular genome and its distribution into daughter cells. Here, the temporal order of these cell cycle events must be strictly enforced-for example, to ensure that DNA replication is c- plete and occurs only once before cell division, or to monitor mitotic spindle assembly, and to prevent exit from mitosis until chromosome segregation has been completed. Thus, well functioning checkpoint mechanisms are central to the maintenance of genomic integrity and the basic viability of cells and, the- fore, are essential for proper development and survival. The importance of proper functioning of checkpoints becomes plainly obvious under conditions in which this control network malfunctions and fails. Depending on the severity and timing, failure of this machinery can lead to embryonic lethality, genetic diseases, and cancer.
List of contents
"Part I. Protocols for the Study of Checkpoint-Regulatory Components
Analysis of RB Action in DNA Damage Checkpoint Response
Christopher N. Mayhew, Emily E. Bosco, David A. Solomon, Erik S. Knudsen, and Steven P. Angus
Interaction Between the Retinoblastoma Protein and Protein Phosphatase 1 During the Cell Cycle
Norbert Berndt and John W. Ludlow
Generation of p53 Target Database Via Integration of Microarray and Global p53 DNA-Binding Site Analysis
Suxing Liu, Asra Mirza, and Luquan Wang
Functional Analysis of CDK Inhibitor p21WAF1
Rati Fotedar, Mourad Bendjennat, and Arun Fotedar
Analysis of p21CDKN1A Recruitment to DNA Excision Repair Foci in the UV-Induced DNA Damage Response
Lucia A. Stivala and Ennio Prosperi
Quantitative Determination of p16 Gene Expression by RT-PCR
Sylke Schneider, Kazumi Uchida, Dennis Salonga, Ji Min Yochim, Kathleen D. Danenberg, and Peter V. Danenberg
Measuring Cyclin-Dependent Kinase Activity
Axel H. Schönthal
Determination of the Catalytic Activities of mTOR and Other Members of the Phosphoinositide-3-Kinase-Related Kinase Family
Gary G. Chiang and Robert T. Abraham
CHK1 Kinase Activity Assay
Ya Wang and Hongyan Wang
Assaying Cdc25 Phosphatase Activity
Ingo Hassepass and Ingrid Hoffmann
Analyzing the Regulation and Function of ATM
Martin F. Lavin, Shaun P. Scott, Sergei Kozlov, and Nuri Gueven
Use of siRNA to Study the Function of MDC1 in DNA Damage Responses
Zhenkun Lou and Junjie Chen
Functional Analysis of APC-Cdh1
Tamotsu Sudo, Naoto T. Ueno, and Hideyuki Saya
Purification of Mitotic Checkpoint Complex, an Inhibitor of the APC/C From HeLa Cells
Valery Sudakin and Tim J. Yen
Analysis of the Spindle-Assembly Checkpoint in HeLa Cells
Paul R. Andreassen, Dimitrios A. Skoufias, and Robert L. MargolisB"
Summary
Intracellular checkpoint controls constitute a network of signal transd- tion pathways that protect cells from external stresses and internal errors. Ext- nal stresses can be generated by the continuous assault of DNA-damaging agents, such as environmental mutagens, ultraviolet (UV) light, ionizing radiation, or the reactive oxygen species that can arise during normal cellular metabolism. In response to any of these assaults on the integrity of the genome, the activation of the network of checkpoint control pathways can lead to diverse cellular responses, such as cell cycle arrest, DNA repair, or elimination of the cell by cell death (apoptosis) if the damage cannot be repaired. Moreover, internal errors can occur during the highly orchestrated replication of the cellular genome and its distribution into daughter cells. Here, the temporal order of these cell cycle events must be strictly enforced—for example, to ensure that DNA replication is c- plete and occurs only once before cell division, or to monitor mitotic spindle assembly, and to prevent exit from mitosis until chromosome segregation has been completed. Thus, well functioning checkpoint mechanisms are central to the maintenance of genomic integrity and the basic viability of cells and, the- fore, are essential for proper development and survival. The importance of proper functioning of checkpoints becomes plainly obvious under conditions in which this control network malfunctions and fails. Depending on the severity and timing, failure of this machinery can lead to embryonic lethality, genetic diseases, and cancer.
