The nuclear kinesin KIF18B promotes 53BP1-mediated DNA double-strand break repair.

53BP1 is recruited to chromatin in the vicinity of DNA double-strand breaks (DSBs). We identify the nuclear kinesin, KIF18B, as a 53BP1-interacting protein and define its role in 53BP1-mediated DSB repair. KIF18B is a molecular motor protein involved in destabilizing astral microtubules during mitosis. It is primarily nuclear throughout the interphase and is constitutively chromatin bound. Our observations indicate a nuclear function during the interphase for a kinesin previously implicated in mitosis. We identify a central motif in KIF18B, which we term the Tudor-interacting motif (TIM), because of its interaction with the Tudor domain of 53BP1. TIM enhances the interaction between the 53BP1 Tudor domain and dimethylated lysine 20 of histone H4. TIM and the motor function of KIF18B are both required for efficient 53BP1 focal recruitment in response to damage and for fusion of dysfunctional telomeres. Our data suggest a role for KIF18B in efficient 53BP1-mediated end-joining of DSBs.

A role for the p53 tumour suppressor in regulating the balance between homologous recombination and non-homologous end joining.

Loss of p53, a transcription factor activated by cellular stress, is a frequent event in cancer. The role of p53 in tumour suppression is largely attributed to cell fate decisions. Here, we provide evidence supporting a novel role for p53 in the regulation of DNA double-strand break (DSB) repair pathway choice. 53BP1, another tumour suppressor, was initially identified as p53 Binding Protein 1, and has been shown to inhibit DNA end resection, thereby stimulating non-homologous end joining (NHEJ). Yet another tumour suppressor, BRCA1, reciprocally promotes end resection and homologous recombination (HR). Here, we show that in both human and mouse cells, the absence of p53 results in impaired 53BP1 focal recruitment to sites of DNA damage induced by ionizing radiation. This effect is largely independent of cell cycle phase and the extent of DNA damage. In p53-deficient cells, diminished localization of 53BP1 is accompanied by a reciprocal increase in BRCA1 recruitment to DSBs. Consistent with these findings, we demonstrate that DSB repair via NHEJ is abrogated, while repair via homology-directed repair (HDR) is stimulated. Overall, we propose that in addition to its role as an 'effector' protein in the DNA damage response, p53 plays a role in the regulation of DSB repair pathway choice.

ATR activates the S-M checkpoint during unperturbed growth to ensure sufficient replication prior to mitotic onset.

Cells must accurately replicate and segregate their DNA once per cell cycle in order to successfully transmit genetic information. During S phase in the presence of agents that cause replication stress, ATR-dependent checkpoints regulate origin firing and the replication machinery as well as prevent untimely mitosis. Here, we investigate the role of ATR during unperturbed growth in vertebrate cells. In the absence of ATR, individual replication forks progress more slowly, and an increased number of replication origins are activated. These cells also enter mitosis early and divide more rapidly, culminating in chromosome bridges and laggards at anaphase, failed cytokinesis, and cell death. Interestingly, cell death can be rescued by prolonging mitosis with partial inhibition of the mitotic cyclin-dependent kinase 1. Our data indicate that one of the essential roles of ATR during normal growth is to minimize the level of unreplicated DNA before the onset of mitosis.

Site-specific phosphorylation of the DNA damage response mediator rad9 by cyclin-dependent kinases regulates activation of checkpoint kinase 1.

The mediators of the DNA damage response (DDR) are highly phosphorylated by kinases that control cell proliferation, but little is known about the role of this regulation. Here we show that cell cycle phosphorylation of the prototypical DDR mediator Saccharomyces cerevisiae Rad9 depends on cyclin-dependent kinase (CDK) complexes. We find that a specific G2/M form of Cdc28 can phosphorylate in vitro the N-terminal region of Rad9 on nine consensus CDK phosphorylation sites. We show that the integrity of CDK consensus sites and the activity of Cdc28 are required for both the activation of the Chk1 checkpoint kinase and its interaction with Rad9. We have identified T125 and T143 as important residues in Rad9 for this Rad9/Chk1 interaction. Phosphorylation of T143 is the most important feature promoting Rad9/Chk1 interaction, while the much more abundant phosphorylation of the neighbouring T125 residue impedes the Rad9/Chk1 interaction. We suggest a novel model for Chk1 activation where Cdc28 regulates the constitutive interaction of Rad9 and Chk1. The Rad9/Chk1 complex is then recruited at sites of DNA damage where activation of Chk1 requires additional DDR-specific protein kinases.

Eukaryotic DNA damage checkpoint activation in response to double-strand breaks.

Double-strand breaks (DSBs) are the most detrimental form of DNA damage. Failure to repair these cytotoxic lesions can result in genome rearrangements conducive to the development of many diseases, including cancer. The DNA damage response (DDR) ensures the rapid detection and repair of DSBs in order to maintain genome integrity. Central to the DDR are the DNA damage checkpoints. When activated by DNA damage, these sophisticated surveillance mechanisms induce transient cell cycle arrests, allowing sufficient time for DNA repair. Since the term "checkpoint" was coined over 20 years ago, our understanding of the molecular mechanisms governing the DNA damage checkpoint has advanced significantly. These pathways are highly conserved from yeast to humans. Thus, significant findings in yeast may be extrapolated to vertebrates, greatly facilitating the molecular dissection of these complex regulatory networks. This review focuses on the cellular response to DSBs in Saccharomyces cerevisiae, providing a comprehensive overview of how these signalling pathways function to orchestrate the cellular response to DNA damage and preserve genome stability in eukaryotic cells.

Modification of histones by sugar ß-N-acetylglucosamine (GlcNAc) occurs on multiple residues, including histone H3 serine 10, and is cell cycle-regulated.

The monosaccharide, ß-N-acetylglucosamine (GlcNAc), can be added to the hydroxyl group of either serines or threonines to generate an O-linked ß-N-acetylglucosamine (O-GlcNAc) residue (Love, D. C., and Hanover, J. A. (2005) Sci. STKE 2005 312, 1-14; Hart, G. W., Housley, M. P., and Slawson, C. (2007) Nature 446, 1017-1022). This post-translational protein modification, termed O-GlcNAcylation, is reversible, analogous to phosphorylation, and has been implicated in many cellular processes. Here, we present evidence that in human cells all four core histones of the nucleosome are substrates for this glycosylation in the relative abundance H3, H4/H2B, and H2A. Increasing the intracellular level of UDP-GlcNAc, the nucleotide sugar donor substrate for O-GlcNAcylation enhanced histone O-GlcNAcylation and partially suppressed phosphorylation of histone H3 at serine 10 (H3S10ph). Expression of recombinant H3.3 harboring an S10A mutation abrogated histone H3 O-GlcNAcylation relative to its wild-type version, consistent with H3S10 being a site of histone O-GlcNAcylation (H3S10glc). Moreover, O-GlcNAcylated histones were lost from H3S10ph immunoprecipitates, whereas immunoprecipitation of either H3K4me3 or H3K9me3 (active or inactive histone marks, respectively) resulted in co-immunoprecipitation of O-GlcNAcylated histones. We also examined histone O-GlcNAcylation during cell cycle progression. Histone O-GlcNAcylation is high in G(1) cells, declines throughout the S phase, increases again during late S/early G(2), and persists through late G(2) and mitosis. Thus, O-GlcNAcylation is a novel histone post-translational modification regulating chromatin conformation during transcription and cell cycle progression.