The ATM (ataxia-telangiectasia mutated) protein kinase mediates early cellular responses to DNA double-strand breaks (DSBs) generated during metabolic processes or by DNA-damaging agents. ATM deficiency leads to ataxia-telangiectasia, a disease marked by lymphopenia, genomic instability and an increased predisposition to lymphoid malignancies with chromosomal translocations involving lymphocyte antigen receptor loci. ATM activates cell-cycle checkpoints and can induce apoptosis in response to DNA DSBs. However, defects in these pathways of the DNA damage response cannot fully account for the phenotypes of ATM deficiency. Here, we show that ATM also functions directly in the repair of chromosomal DNA DSBs by maintaining DNA ends in repair complexes generated during lymphocyte antigen receptor gene assembly. When coupled with the cell-cycle checkpoint and pro-apoptotic activities of ATM, these findings provide a molecular explanation for the increase in lymphoid tumours with translocations involving antigen receptor loci associated with ataxia-telangiectasia.

Single-strand DNA (ssDNA)-binding proteins (SSBs) are ubiquitous and essential for a wide variety of DNA metabolic processes, including DNA replication, recombination, DNA damage detection and repair. SSBs have multiple roles in binding and sequestering ssDNA, detecting DNA damage, stimulating nucleases, helicases and strand-exchange proteins, activating transcription and mediating protein-protein interactions. In eukaryotes, the major SSB, replication protein A (RPA), is a heterotrimer. Here we describe a second human SSB (hSSB1), with a domain organization closer to the archaeal SSB than to RPA. Ataxia telangiectasia mutated (ATM) kinase phosphorylates hSSB1 in response to DNA double-strand breaks (DSBs). This phosphorylation event is required for DNA damage-induced stabilization of hSSB1. Upon induction of DNA damage, hSSB1 accumulates in the nucleus and forms distinct foci independent of cell-cycle phase. These foci co-localize with other known repair proteins. In contrast to RPA, hSSB1 does not localize to replication foci in S-phase cells and hSSB1 deficiency does not influence S-phase progression. Depletion of hSSB1 abrogates the cellular response to DSBs, including activation of ATM and phosphorylation of ATM targets after ionizing radiation. Cells deficient in hSSB1 exhibit increased radiosensitivity, defective checkpoint activation and enhanced genomic instability coupled with a diminished capacity for DNA repair. These findings establish that hSSB1 influences diverse endpoints in the cellular DNA damage response.

Synthetic lethality is a powerful approach to study selective cell killing based on genotype. We show that loss of Rad52 function is synthetically lethal with breast cancer 2, early onset (BRCA2) deficiency, whereas there was no impact on cell growth and viability in BRCA2-complemented cells. The frequency of both spontaneous and double-strand break-induced homologous recombination and ionizing radiation-induced Rad51 foci decreased by 2-10 times when Rad52 was depleted in BRCA2-deficient cells, with little to no effect in BRCA2-complemented cells. The absence of both Rad52 and BRCA2 resulted in extensive chromosome aberrations, especially chromatid-type aberrations. Ionizing radiation-induced and S phase-associated Rad52-Rad51 foci form equally well in the presence or absence of BRCA2, indicating that Rad52 can respond to DNA double-strand breaks and replication stalling independently of BRCA2. Rad52 thus is an independent and alternative repair pathway of homologous recombination and a target for therapy in BRCA2-deficient cells.DNA repair | genetic instability | chromosomal aberrations D NA double-strand breaks (DSBs) are potentially lethal DNA lesions which may arise spontaneously during DNA replication or result from exposure to ionizing radiation or other DNAdamaging agents (1). To repair DSBs, eukaryotes have developed two DSB repair pathways: nonhomologous end joining and homologous recombination (HR) (2). HR is required for the repair of complex double-strand lesions such as crosslinks or one-ended DSBs that occur with a cleaved replication fork; nonhomologous end joining appears to have little role in the repair of these lesions. The absence of Rad51 in proliferating cells (and therefore any measurable HR) results in cell lethality. The loss of function of proteins involved in HR, such as breast cancer 2, early onset (BRCA2), will be viable only if there is a BRCA2-independent pathway for Rad51 function.In Saccharomyces cerevisiae, the Rad52 protein plays a key role in HR (3). However, in vertebrates, knockouts of the Rad52 gene show little phenotype, with no obvious defect in HR. Rad52 knockout mice exhibit a nearly normal phenotype, and Rad52-deficient embryonic stem cells are not hypersensitive to agents that induce DSBs, either simple or complex (4, 5). In contrast, Rad51 knockout is embryonically lethal (6, 7), and depletion of Rad51 from vertebrate cells results in an accumulation of chromosome aberrations and subsequent cell death (8). These findings indicate the essential role of Rad51 in the maintenance of chromosomal DNA during the mitotic cell cycle, but the role for Rad52 in vertebrate cells is unclear.Accumulating evidence implicates BRCA2 as an integral component of the HR machinery via the direct regulation of the assembly of Rad51 filaments and its subsequent activity in strand exchange (9-11). Biochemical studies showed that the Ustilago maydis BRCA2 ortholog, Brh2, is involved in the recruitment of Rad51 to the sites of HR; Rad51 then mediates the displacement of replication protein A...

The human MOF gene encodes a protein that specifically acetylates histone H4 at lysine 16 (H4K16ac). Here we show that reduced levels of H4K16ac correlate with a defective DNA damage response (DDR) and double-strand break (DSB) repair to ionizing radiation (IR). The defect, however, is not due to altered expression of proteins involved in DDR. Abrogation of IR-induced DDR by MOF depletion is inhibited by blocking H4K16ac deacetylation. MOF was found to be associated with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a protein involved in nonhomologous end-joining (NHEJ) repair. ATM-dependent IR-induced phosphorylation of DNA-PKcs was also abrogated in MOF-depleted cells. Our data indicate that MOF depletion greatly decreased DNA double-strand break repair by both NHEJ and homologous recombination (HR). In addition, MOF activity was associated with general chromatin upon DNA damage and colocalized with the synaptonemal complex in male meiocytes. We propose that MOF, through H4K16ac (histone code), has a critical role at multiple stages in the cellular DNA damage response and DSB repair.In eukaryotes, specifically in mammals, the mechanisms by which the DNA damage response (DDR) components gain access to broken DNA in compacted chromatin remain a mystery. The DNA damage response occurs within the context of chromatin, and its structure is altered post-DNA double-strand break (DSB) induction. Major alterations include (i) chromatin remodeling via ATP-dependent activities and covalent histone modifications and (ii) incorporation of histone variants into nucleosomes. Chromatin structure creates a natural barrier to damaged DNA sites, which suggests that histone modifications will play a primary role in DDR by facilitating repair protein access to DNA breaks (43,58,87,88). While some experimental evidence indicates that preexisting histone modifications may play an important role in DDR, the precise role of chromatin status prior to DNA damage on DDR is yet to be clearly established. For instance, biochemical and cell biology studies indicate that repair proteins (53BP1, Schizosaccharomyces pombe Crb2 [SpCrb2], and Saccharomyces cerevisiae Rad9 [ScRad9]) require methylated Lys79 of histone H3 (H3-K79) (29) or methylated Lys20 of histone H4 (H4-K20) and/or CBP/p300-mediated acetylation of histone H3 on lysine 56 (9,15,29,66,93) for focus formation at DNA-damaged sites. These modifications are normally present on chromatin, and none has been reported to change in response to ionizing radiation (IR)-induced DNA damage. However, it is yet to be established whether preexisting acetylation of specific histone residues at the time of cellular exposure to IR plays any critical role in DDR. While recent studies demonstrate that in human cells, histone H3 acetylated at K9 (H3K9ac) and H3K56ac are rapidly and reversibly reduced in response to DNA damage, most histone acetylation modifications do not change appreciably after genotoxic stress (80).The amino-terminal tail of histone H4 is a well-described target fo...

We have determined that hMOF, the human ortholog of the Drosophila MOF gene (males absent on the first), encoding a protein with histone acetyltransferase activity, interacts with the ATM (ataxia-telangiectasiamutated) protein. Cellular exposure to ionizing radiation (IR) enhances hMOF-dependent acetylation of its target substrate, lysine 16 (K16) of histone H4 independently of ATM function. Blocking the IR-induced increase in acetylation of histone H4 at K16, either by the expression of a dominant negative mutant ⌬hMOF or by RNA interference-mediated hMOF knockdown, resulted in decreased ATM autophosphorylation, ATM kinase activity, and the phosphorylation of downstream effectors of ATM and DNA repair while increasing cell killing. In addition, decreased hMOF activity was associated with loss of the cell cycle checkpoint response to DNA double-strand breaks. The overexpression of wild-type hMOF yielded the opposite results, i.e., a modest increase in cell survival and enhanced DNA repair after IR exposure. These results suggest that hMOF influences the function of ATM.In eukaryotic cells, DNA damage activates signal transduction pathways that rapidly affect downstream processes such as gene transcription, cell cycle progression, and DNA replication (13,25). All of these processes involve alterations in chromatin structure. Posttranslational covalent modifications of histones have emerged as key regulatory events in DNA damage response. A widespread modification is acetylation catalyzed by histone acetyltransferases and reversed by deacetylases (3, 13, 50). Reversible acetylation of four lysines (K) at positions 5, 8, 12, and 16 in the amino-terminal tail of histone H4 occurs in vivo in all eukaryotes (3). The hyperacetylation of histone H4 could lead to the unfolding of the nucleosomal fiber (50), and the acetylation of histone H4 at K16 occurs on the hyperactive male X chromosome of Drosophila polytene chromosomes (51). Ikura et al. (19) noted that Tip60 (Tat-interacting protein), which acetylates histones H2A, H3, and H4, plays a role in DNA repair. More recently, Kusch et al. (28) demonstrated that the Drosophila Tip60 acetylates nucleosomal phosphoH2Av and exchanges it with an unmodified H2Av. Bird et al. (5) reported that the acetylation of histone H4 by Esa1 (essential SAS2-related acetyltransferase) is required for DNA repair in yeast and suggested that a similar modification may function in mammalian cells.ATM (ataxia-telangiectasia-mutated protein) is crucial for the initiation of signaling pathways in mammalian cells following exposure to ionizing radiation (IR) and other DNA-damaging agents (36, 46), and cells deficient in ATM function also have defective telomere chromatin (47). Bakkenist and Kastan (4) have suggested that chromatin structural perturbations induced by DNA double-strand breaks (DSBs) serve as a trigger for ATM activation. Recent studies indicate that the MRN (Mre11, Rad50, and Nbs1) complex as well as TRF2 either influences activation of ATM (9, 29, 52) or serves as a modulator/amp...