In mammalian cells, repair of DNA double-strand breaks (DSBs) by nonhomologous end-joining (NHEJ) is critical for genome stability. Although the end-bridging and ligation steps of NHEJ have been reconstituted in vitro, little is known about the end-processing reactions that occur before ligation. Recently, functionally homologous end-bridging and ligation activities have been identified in prokarya. Consistent with its homology to polymerases and nucleases, we demonstrate that DNA ligase D from Mycobacterium tuberculosis (Mt-Lig) possesses a unique variety of nucleotidyl transferase activities, including gap-filling polymerase, terminal transferase, and primase, and is also a 3' to 5' exonuclease. These enzyme activities allow the Mt-Ku and Mt-Lig proteins to join incompatible DSB ends in vitro, as well as to reconstitute NHEJ in vivo in yeast. These results demonstrate that prokaryotic Ku and ligase form a bona fide NHEJ system that encodes all the recognition, processing, and ligation activities required for DSB repair.

Nonhomologous end joining (NHEJ) is an important DNA double-strand-break (DSB) repair pathway that requires three protein complexes in Saccharomyces cerevisiae: the Ku heterodimer (Yku70-Yku80), MRX (Mre11-Rad50-Xrs2), and DNA ligase IV (Dnl4-Lif1), as well as the ligase-associated protein Nej1. Here we use chromatin immunoprecipitation from yeast to dissect the recruitment and release of these protein complexes at HO-endonuclease-induced DSBs undergoing productive NHEJ. Results revealed that Ku and MRX assembled at a DSB independently and rapidly after DSB formation. Ligase IV appeared at the DSB later than Ku and MRX and in a strongly Ku-dependent manner. Ligase binding was extensive but slightly delayed in rad50 yeast. Ligase IV binding occurred independently of Nej1, but instead promoted loading of Nej1. Interestingly, dissociation of Ku and ligase from unrepaired DSBs depended on the presence of an intact MRX complex and ATP binding by Rad50, suggesting a possible role of MRX in terminating a NHEJ repair phase. This activity correlated with extended DSB resection, but limited degradation of DSB ends occurred even in MRX mutants with persistently bound Ku. These findings reveal the in vivo assembly of the NHEJ repair complex and shed light on the mechanisms controlling DSB repair pathway utilization.

The ubiquitin-dependent proteolysis of mitotic cyclin B, which is catalyzed by the anaphase-promoting complex/cyclosome (APC/C) and ubiquitin-conjugating enzyme H10 (UbcH10), begins around the time of the metaphase-anaphase transition and continues through G1 phase of the next cell cycle. We have used cell-free systems from mammalian somatic cells collected at different cell cycle stages (G0, G1, S, G2, and M) to investigate the regulated degradation of four targets of the mitotic destruction machinery: cyclins A and B, geminin H (an inhibitor of S phase identified in Xenopus), and Cut2p (an inhibitor of anaphase onset identified in fission yeast). All four are degraded by G1 extracts but not by extracts of S phase cells. Maintenance of destruction during G1 requires the activity of a PP2A-like phosphatase. Destruction of each target is dependent on the presence of an N-terminal destruction box motif, is accelerated by additional wild-type UbcH10 and is blocked by dominant negative UbcH10. Destruction of each is terminated by a dominant activity that appears in nuclei near the start of S phase. Previous work indicates that the APC/C-dependent destruction of anaphase inhibitors is activated after chromosome alignment at the metaphase plate. In support of this, we show that addition of dominant negative UbcH10 to G1 extracts blocks destruction of the yeast anaphase inhibitor Cut2p in vitro, and injection of dominant negative UbcH10 blocks anaphase onset in vivo. Finally, we report that injection of dominant negative Ubc3/Cdc34, whose role in G1-S control is well established and has been implicated in kinetochore function during mitosis in yeast, dramatically interferes with congression of chromosomes to the metaphase plate. These results demonstrate that the regulated ubiquitination and destruction of critical mitotic proteins is highly conserved from yeast to humans.

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Cdc34/Ubc3 is a ubiquitin-conjugating enzyme that functions in targeting proteins for proteasome-mediated degradation at the G1 to S cell cycle transition. Elevation of Cdc34 protein levels by microinjection of bacterially expressed Cdc34 into mammalian cells at prophase inhibited chromosome congression to the metaphase plate with many chromosomes remaining near the spindle poles. Chromosome condensation and nuclear envelope breakdown occurred normally, and chromosomes showed oscillatory movements along mitotic spindle microtubules. Most injected cells arrested in a prometaphase-like state. Kinetochores, even those of chromosomes that failed to congress, possessed the normal trilaminar plate ultrastructure. The elevation of Cdc34 protein levels in early mitosis selectively blocked centromere protein E (CENP-E), a mitotic kinesin, from associating with kinetochores. Other proteins, including two CENP-E–associated proteins, BubR1 and phospho-p42/p44 mitogen-activated protein kinase, and mitotic centromere-associated kinesin, cytoplasmic dynein, Cdc20, and Mad2, all exhibited normal localization to kinetochores. Proteasome inhibitors did not affect the prometaphase arrest induced by Cdc34 injection. These studies suggest that CENP-E targeting to kinetochores is regulated by ubiquitylation not involving proteasome-mediated degradation.

science. They want to be excited, to be wowed by the applications and by the discussions that they engender -that is their intellectual payoff for the hard work and angst engendered by study section service. Reviewers do not begin by looking for reasons to denigrate an application; they look for substance, for reasons to say, 'this work must be done -if successful it will surely have a great impact.' Much is said about the conservatism of study sections but in my experience this is mainly a reflection of the conservatism of applicants.Most applications are distressingly similar. The organisms or systems may change but the questions and approaches barely differ. A novel application, well argued, stands out like a bright beacon and is rewarded by study section members.There is another important factor that motivates study section members to be fair. Participating in the meeting are some of the influential practitioners in their field who know who is competing and who is collaborating. These are colleagues whose respect it is imperative study section members maintain. The upshot is that reviewers give their competitors the benefit of the doubt and collaborators are held to a very high standard. Few wish to look the fool by seeming to be selfserving or gullible.The NIH peer review system is not perfect, nor do I believe that it is perfectible since it depends to such a high degree on the personalities and talents of people. It is, as many have said, the best system we have for distributing the largesse provided by the public. It works best when all participants recognize that it is a human endeavor with all the strengths and weaknesses that that entails.

The spindle checkpoint blocks the initiation of anaphase in mitosis and meiosis if chromosomes are not aligned at the metaphase plate. The checkpoint functions by preventing a ubiquitin ligase called the anaphase‐promoting complex/cy‐closome (APC/C) from ubiquitinylating proteins whose destruction is required for anaphase onset. The spindle checkpoint signal originates at the kinetochores of unaligned chromosomes and is broadcast to the rest of the cell. Although the spindle checkpoint is not understood in detail, several components of the checkpoint‐signaling pathway have been identified. Many of these components associate transiently with the kinetochores of unaligned chromosomes. We propose a model in which kinetochores that lack stable attachments to the spindle microtubules serve as catalytic staging areas for the assembly of inhibitor complexes. These inhibitor complexes then leave the kinetochores and block activity of the APC/C throughout the cell. We suggest that microtubule occupancy at kinetochores or physical tension induced by microtubule capture turns off the capability of the kinetochore to produce the APC/C inhibitor. Subsequently, the inhibitor concentration in the cell wanes and anaphase initiates.—Gorbsky, G. J., Kallio, M., Daum, J. R., Topper, L. M. Protein dynamics at the kinetochore: cell cycle regulation of the metaphase to anaphase transition. FASEB J. 13 (Suppl.), S231‐S234 (1999)