James E. Haber

DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1

A single double-strand break (DSB) induced by HO endonuclease triggers both repair by homologous recombination and activation of the Mec1-dependent DNA damage checkpoint in budding yeast. Here we report that DNA damage checkpoint activation by a DSB requires the cyclin-dependent kinase CDK1 (Cdc28) in budding yeast. CDK1 is also required for DSB-induced homologous recombination at any cell cycle stage. Inhibition of homologous recombination by using an analogue-sensitive CDK1 protein results in a compensatory increase in non-homologous end joining. CDK1 is required for efficient 5′ to 3′ resection of DSB ends and for the recruitment of both the single-stranded DNA-binding complex, RPA, and the Rad51 recombination protein. In contrast, Mre11 protein, part of the MRX complex, accumulates at unresected DSB ends. CDK1 is not required when the DNA damage checkpoint is initiated by lesions that are processed by nucleotide excision repair. Maintenance of the DSB-induced checkpoint requires continuing CDK1 activity that ensures continuing end resection. CDK1 is also important for a later step in homologous recombination, after strand invasion and before the initiation of new DNA synthesis.

Partners and pathways: repairing a double-strand break

Double-strand chromosome breaks can arise in a number of ways, by ionizing radiation, by spontaneous chromosome breaks during DNA replication, or by the programmed action of endonucleases, such as in meiosis. Broken chromosomes can be repaired either by one of several homologous recombination mechanisms, or by a number of nonhomologous repair processes. Many of these pathways compete actively for the repair of a double-strand break. Which of these repair pathways is used appears to be regulated developmentally, genetically and during the cell cycle.

INO80 and γ-H2AX Interaction Links ATP-Dependent Chromatin Remodeling to DNA Damage Repair

While the role of ATP-dependent chromatin remodeling in transcription is well established, a link between chromatin remodeling and DNA repair has remained elusive. We have found that the evolutionarily conserved INO80 chromatin remodeling complex directly participates in the repair of a double-strand break (DSB) in yeast. The INO80 complex is recruited to a HO endonuclease-induced DSB through a specific interaction with the DNA damage-induced phosphorylated histone H2A (γ-H2AX). This interaction requires Nhp10, an HMG-like subunit of the INO80 complex. The loss of Nhp10 or γ-H2AX results in reduced INO80 recruitment to the DSB. Finally, components of the INO80 complex show synthetic genetic interactions with the RAD52 DNA repair pathway, the main pathway for DSB repair in yeast. Our findings reveal a new role of ATP-dependent chromatin remodeling in nuclear processes and suggest that an ATP-dependent chromatin remodeling complex can read a DNA repair histone code.

Srs2 and Sgs1–Top3 Suppress Crossovers during Double-Strand Break Repair in Yeast

Very few gene conversions in mitotic cells are associated with crossovers, suggesting that these events are regulated. This may be important for the maintenance of genetic stability. We have analyzed the relationship between homologous recombination and crossing-over in haploid budding yeast and identified factors involved in the regulation of crossover outcomes. Gene conversions unaccompanied by a crossover appear 30 min before conversions accompanied by exchange, indicating that there are two different repair mechanisms in mitotic cells. Crossovers are rare (5%), but deleting the BLM/WRN homolog, SGS1, or the SRS2 helicase increases crossovers 2- to 3-fold. Overexpressing SRS2 nearly eliminates crossovers, whereas overexpression of RAD51 in srs2Delta cells almost completely eliminates the noncrossover recombination pathway. We suggest Sgs1 and its associated topoisomerase Top3 remove double Holliday junction intermediates from a crossover-producing repair pathway, thereby reducing crossovers. Srs2 promotes the noncrossover synthesis-dependent strand-annealing (SDSA) pathway, apparently by regulating Rad51 binding during strand exchange.

Mating-type gene switching in Saccharomyces cerevisiae

The study of yeast mating-type (MAT) gene switching has provided insights into several aspects of the regulation of gene expression. MAT switching is accomplished by a highly programmed site-specific homologous recombination event in which mating-type-specific sequences at MAT are replaced by alternative DNA sequences copied from one of two unexpressed donors. The mating-type system has also provided an opportunity to study both the genetic regulation of gene silencing by alterations in chromatin structure, and the basis of preferential recombination between a recipient of genetic information and one of several possible donors.

Distribution and Dynamics of Chromatin Modification Induced by a Defined DNA Double-Strand Break

BACKGROUND In response to DNA double-strand breaks (DSBs), eukaryotic cells rapidly phosphorylate histone H2A isoform H2AX at a C-terminal serine (to form gamma-H2AX) and accumulate repair proteins at or near DSBs. To date, these events have been defined primarily at the resolution of light microscopes, and the relationship between gamma-H2AX formation and repair protein recruitment remains to be defined. RESULTS We report here the first molecular-level characterization of regional chromatin changes that accompany a DSB formed by the HO endonuclease in Saccharomyces cerevisiae. Break induction provoked rapid gamma-H2AX formation and equally rapid recruitment of the Mre11 repair protein. gamma-H2AX formation was efficiently promoted by both Tel1p and Mec1p, the yeast ATM and ATR homologs; in G1-arrested cells, most gamma-H2AX formation was dependent on Tel1 and Mre11. gamma-H2AX formed in a large (ca. 50 kb) region surrounding the DSB. Remarkably, very little gamma-H2AX could be detected in chromatin within 1-2 kb of the break. In contrast, this region contains almost all the Mre11p and other repair proteins that bind as a result of the break. CONCLUSIONS Both Mec1p and Tel1p can respond to a DSB, with distinct roles for these checkpoint kinases at different phases of the cell cycle. Part of this response involves histone phosphorylation over large chromosomal domains; however, the distinct distributions of gamma-H2AX and repair proteins near DSBs indicate that localization of repair proteins to breaks is not likely to be the main function of this histone modification.

The Many Interfaces of Mre11

This year, the Mre11 complex has attracted a new group of aficionados interested in cancer and in checkpoint regulation. In humans, the p95 component is mutated in Nijmegen breakage syndrome (NBS), a condition that has similarities with ataxia telangiectasia, including ionizing radiation-sensitivity, cancer predisposition, and a failure to arrest at G1/S in response to DNA damage (5xCarney, J.P, Maser, R.S, Olivares, H, Davis, E.M, Le Beau, M, Yates, J.R III, Hays, L, Morgan, W.F, and Petrini, J.H. Cell. 1998; 93: 477–486Abstract | Full Text | Full Text PDF | PubMed | Scopus (858)See all References, 26xVaron, R, Vissinga, C, Platzer, M, Cerosaletti, K.M, Chrzanowska, K.H, Saar, K, Beckmann, G, Seemanova, E, Cooper, P.R, Nowak, N.J et al. Cell. 1998; 93: 467–476Abstract | Full Text | Full Text PDF | PubMed | Scopus (725)See all References). Possibly, Mre11/Rad50/NBS binds to DSB ends and signals the presence of that damage.An alternative possibility is that the signal of DNA damage is the extent of single-stranded DNA produced by the exonuclease activity of the Mre11 complex. In Saccharomyces, evidence supporting this idea has come not from studying the ability of cells to arrest after DNA damage, but from their capacity to adapt and resume growth when damage persists. Two DSBs are sufficient to discourage a wild-type yeast cell from adapting to checkpoint-induced G2/M arrest, but this permanent arrest is suppressed by an mre11 or rad50 deletion that reduces the extent of single-stranded DNA (Lee et al. 1998xLee, S.-E, Moore, J.K, Holmes, A, Umezu, K, Kolodner, R, and Haber, J.E. Cell. 1998; 94: 399–409Abstract | Full Text | Full Text PDF | PubMed | Scopus (520)See all ReferencesLee et al. 1998).There is no doubt that Mre11p is attracted to the sites of DNA damage, for repair and possibly as part of the damage-signaling apparatus. Following irradiation, foci containing both Mre11p and Rad50p have been seen in mammalian cells and in yeast. A stunning series of micrographs illustrating this point were published by Nelms et al. 1998xNelms, B.E, Maser, R.S, MacKay, J.F, Lagally, M.G, and Petrini, J.H. Science. 1998; 280: 590–592Crossref | PubMed | Scopus (376)See all ReferencesNelms et al. 1998, who examined mammalian nuclei irradiated with ultrasoft X-rays passed through a grid that produced stripes of DNA damage. The remarkable finding was not that hMre11p was localized within the irradiated regions, but that in repair-defective cells these stripes persisted for hours, suggesting that the damaged DNA was not diffusing around the nucleus. Interestingly, these foci do not attract Rad51p. If DNA ends were being resected, then the 3′-ended single-stranded regions ought to be attractive sites for the assembly of Rad51p filaments that is the first step in repairing DSBs by homologous recombination. However, no such foci containing both hMre11p and hRad51p were seen. A similar lack of colocalization has been seen in yeast cells that cannot complete meiosis and in irradiated mitotic cells (Usui et al. 1998xUsui, T, Ohta, T, Oshiumi, H, Tomizawa, J, Ogawa, H, and Ogawa, T. Cell. 1998; 95: 705–716Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesUsui et al. 1998).The Mre11 complex is attracted to DSBs and can participate in several different pathways of repair and recombination. It remains “only” to figure out how Mre11p, Rad50p, Xrs2p, and the proteins with which they interact enable the cell to perform these many tasks. Given that almost half the papers on Mre11p have been published in 1998, it is likely we will soon learn much more about the many facets of this multitalented protein.

A phosphatase complex that dephosphorylates γH2AX regulates DNA damage checkpoint recovery

One of the earliest marks of a double-strand break (DSB) in eukaryotes is serine phosphorylation of the histone variant H2AX at the carboxy-terminal SQE motif to create γH2AX-containing nucleosomes. Budding-yeast histone H2A is phosphorylated in a similar manner by the checkpoint kinases Tel1 and Mec1 (ref. 2; orthologous to mammalian ATM and ATR, respectively) over a 50-kilobase region surrounding the DSB. This modification is important for recruiting numerous DSB-recognition and repair factors to the break site, including DNA damage checkpoint proteins, chromatin remodellers and cohesins. Multiple mechanisms for eliminating γH2AX as DNA repair completes are possible, including removal by histone exchange followed potentially by degradation, or, alternatively, dephosphorylation. Here we describe a three-protein complex (HTP-C, for histone H2A phosphatase complex) containing the phosphatase Pph3 that regulates the phosphorylation status of γH2AX in vivo and efficiently dephosphorylates γH2AX in vitro. γH2AX is lost from chromatin surrounding a DSB independently of the HTP-C, indicating that the phosphatase targets γH2AX after its displacement from DNA. The dephosphorylation of γH2AX by the HTP-C is necessary for efficient recovery from the DNA damage checkpoint.

DNA recombination: the replication connection

Chromosomal double-strand breaks (DSBs) arise after exposure to ionizing radiation or enzymatic cleavage, but especially during the process of DNA replication itself. Homologous recombination plays a critical role in repair of such DSBs. There has been significant progress in our understanding of two processes that occur in DSB repair: gene conversion and recombination-dependent DNA replication. Recent evidence suggests that gene conversion and break-induced replication are related processes that both begin with the establishment of a replication fork in which both leading- and lagging-strand synthesis occur. There has also been much progress in characterization of the biochemical roles of recombination proteins that are highly conserved from yeast to humans.

Telomere maintenance is dependent on activities required for end repair of double-strand breaks

Telomeres are functionally distinct from ends generated by chromosome breakage, in that telomeres, unlike double-strand breaks, are insulated from recombination with other chromosomal termini [1]. We report that the Ku heterodimer and the Rad50/Mre11/Xrs2 complex, both of which are required for repair of double-strand breaks [2-5], have separate roles in normal telomere maintenance in yeast. Using epistasis analysis, we show that the Ku end-binding complex defined a third telomere-associated activity, required in parallel with telomerase [6] and Cdc13, a protein binding the single-strand portion of telomere DNA [7,8]. Furthermore, loss of Ku function altered the expression of telomere-located genes, indicative of a disruption of telomeric chromatin. These data suggest that the Ku complex and the Cdc13 protein function as terminus-binding factors, contributing distinct roles in chromosome end protection. In contrast, MRE11 and RAD50 were required for the telomerase-mediated pathway, rather than for telomeric end protection; we propose that this complex functions to prepare DNA ends for telomerase to replicate. These results suggest that as a part of normal telomere maintenance, telomeres are identified as double-strand breaks, with additional mechanisms required to prevent telomere recombination. Ku, Cdc13 and telomerase define three epistasis groups required in parallel for telomere maintenance.