Full Description
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.
Contents
Protocols for the Study of Checkpoint Regulatory Components.- Analysis of RB Action in DNA Damage Checkpoint Response.- Interaction Between the Retinoblastoma Protein and Protein Phosphatase 1 During the Cell Cycle.- Generation of p53 Target Database Via Integration of Microarray and Global p53 DNA-Binding Site Analysis.- Functional Analysis of CDK Inhibitor p21WAF1.- Analysis of p21CDKN1A Recruitment to DNA Excision Repair Foci in the UV-Induced DNA Damage Response.- Quantitative Determination of p16 Gene Expression by RT-PCR.- Measuring Cyclin-Dependent Kinase Activity.- Determination of the Catalytic Activities of mTOR and Other Members of the Phosphoinositide-3-Kinase-Related Kinase Family.- CHK1 Kinase Activity Assay.- Assaying Cdc25 Phosphatase Activity.- Analyzing the Regulation and Function of ATM.- Use of siRNA to Study the Function of MDC1 in DNA Damage Responses.- Functional Analysis of APC-Cdh1.- Purification of the Mitotic Checkpoint Complex, an Inhibitor of the APC/C From HeLa Cells.- Analysis of the Spindle-Assembly Checkpoint in HeLa Cells.- Functional Analysis of the Spindle-Checkpoint Proteins Using an In Vitro Ubiquitination Assay.- Studying Consequences of Checkpoint Pathway Activation.- Analysis of Checkpoint Responses to Histone Deacetylase Inhibitors.- Biochemical Analysis of the Cell Cycle and Cell Cycle Checkpoints in Transiently Transfected Cells After Collection With Magnetic Beads.- Analysis of DNA Repair and Chromatin Assembly In Vitro Using Immobilized Damaged DNA Substrates.- Analyzing Cell Cycle Checkpoints After Ionizing Radiation.- FACS-Based Detection of Phosphorylated Histone H3 for the Quantitation of Mitotic Cells.- Analysis of Cell Cycle by Flow Cytometry.- Analyzing Markers of Apoptosis In Vitro.- Analysis of Telomerase Activity and Telomere Function in Cancer.
