Cell-cycle transitions in higher eukaryotes are regulated by different cyclin-dependent kinases (CDKs) and their activating cyclin subunits. Based on pioneering findings that a dominant-negative mutation of CDK1 blocks the cell cycle at G2-M phase, whereas dominant-negative CDK2 inhibits the transition into S phase, a model of cell-cycle control has emerged in which each transition is regulated by a specific subset of CDKs and cyclins. Recent work with gene-targeted mice has led to a revision of this model. We discuss cell-cycle control in light of overlapping and essential functions of the different CDKs and cyclins.

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Single-strand breaks are the commonest lesions arising in cells, and defects in their repair are implicated in neurodegenerative disease. One of the earliest events during single-strand break repair (SSBR) is the rapid synthesis of poly(ADP-ribose) (PAR) by poly(ADP-ribose) polymerase (PARP), followed by its rapid degradation by poly(ADP-ribose) glycohydrolase (PARG). While the synthesis of poly(ADP-ribose) is important for rapid rates of chromosomal SSBR, the relative importance of poly(ADP-ribose) polymerase 1 (PARP-1) and PARP-2 and of the subsequent degradation of PAR by PARG is unclear. Here we have quantified SSBR rates in human A549 cells depleted of PARP-1, PARP-2, and PARG, both separately and in combination. We report that whereas PARP-1 is critical for rapid global rates of SSBR in human A549 cells, depletion of PARP-2 has only a minor impact, even in the presence of depleted levels of PARP-1. Moreover, we identify PARG as a novel and critical component of SSBR that accelerates this process in concert with PARP-1.Single-strand breaks (SSBs) are the commonest type of lesion arising in cells and can arise from direct attack of deoxyribose, as abortive intermediates of topoisomerase 1 activity, or as normal intermediates of base excision repair. One of the earliest responses to DNA strand breakage is the induction of poly(ADP-ribose) (PAR) synthesis (reviewed in references 17 and 35). Poly(ADP-ribose) polymerase 1 (PARP-1) is an abundant and stable component of chromatin and is the major source of PAR synthesis following DNA strand breakage (27,40). PARP-1 rapidly binds to and is activated by DNA singleand double-strand breaks, resulting in covalent modification of itself and to a lesser extent other target proteins with long chains of PAR (4,5,15,41,42). The binding and activity of PARP-1 at DNA breaks are very transient because the ribosylated enzyme dissociates from DNA through charge repulsion (24, 60). Subsequently, a second DNA damage-activated PARP was identified in human cells and was called PARP-2 (1, 30). PARP-2 has 18-fold lower activity than PARP-1 but can support up to 25% of normal levels of DNA damage-induced PAR synthesis in the absence of PARP-1 (1, 49). While PARP-1 is the primary source of global PAR synthesis following DNA strand breakage, it is possible that PARP-2 fulfils an overlapping or backup role. In support of this, mice lacking either PARP-1 or PARP-2 are viable, but mice lacking both enzymes are not (38). The presence of high levels of PAR in cells following DNA strand breakage is very transient because the polymer is rapidly degraded by poly(ADP-ribose) glycohydrolase (PARG). Consequently, proteins that become ribosylated following DNA strand breakage are rapidly converted back to their unmodified form (16,32,56,60). PARG is composed of a 110-kDa nuclear form and at least two cytoplasmic isoforms of 99 kDa and 103 kDa, each of which most likely arises from the same primary transcript (34, 39).Despite their central roles in PAR metabolism, the relative importance of PARP-1, ...

Centromeres form the site of chromosome attachment to microtubules during mitosis. Identity of these loci is maintained epigenetically by nucleosomes containing the histone H3 variant CENP-A. Propagation of CENP-A chromatin is uncoupled from DNA replication initiating only during mitotic exit. We now demonstrate that inhibition of Cdk1 and Cdk2 activities is sufficient to trigger CENP-A assembly throughout the cell cycle in a manner dependent on the canonical CENP-A assembly machinery. We further show that the key CENP-A assembly factor Mis18BP1(HsKNL2) is phosphorylated in a cell cycle-dependent manner that controls its centromere localization during mitotic exit. These results strongly support a model in which the CENP-A assembly machinery is poised for activation throughout the cell cycle but kept in an inactive noncentromeric state by Cdk activity during S, G2, and M phases. Alleviation of this inhibition in G1 phase ensures tight coupling between DNA replication, cell division, and subsequent centromere maturation.

Parp-1 and Parp-2 are activated by DNA breaks and have been implicated in the repair of DNA single-strand breaks (SSB). Their involvement in double-strand break (DSB) repair mediated by homologous recombination (HR) or nonhomologous end joining (NHEJ) remains unclear. We addressed this question using chicken DT40 cells, which have the advantage of carrying only a PARP-1 gene but not a PARP-2 gene. We found that PARP-1 À/À DT40 mutants show reduced levels of HR and are sensitive to various DSB-inducing genotoxic agents. Surprisingly, this phenotype was strictly dependent on the presence of Ku, a DSBbinding factor that mediates NHEJ. PARP-1/KU70 double mutants were proficient in the execution of HR and displayed elevated resistance to DSB-inducing drugs. Moreover, we found deletion of Ligase IV, another NHEJ gene, suppressed the camptothecin of PARP-1 À/À cells. Our results suggest a new critical function for Parp in minimizing the suppressive effects of Ku and the NHEJ pathway on HR.