Binding of phosphorylated histone H1 to DNA.

A chromatin associated protein kinase was used to add 3 moles of phosphate to seryl side chains of 1 mole of histone H1. The DNA binding properties of this in vitro phosphorylated H1 were compared with those of unmodified H1. Considerably more radioactive superhelical DNA was retained on nitrocellulose filters at 20mM-40mM NaCl by phosphorylated H1 than by unmodified H1. However, zone velocity sedimentation analysis of histone-DNA complexes indicated that similar amounts of phosphorylated and unmodified H1 are bound to DNA. It is therefore concluded that phosphorylated H1 binds distributively to many or all DNA molecules available (depending on the histone/DNA ratio) while unmodified H1 binds cooperatively to a fraction of the DNA molecules in the reaction mixture.     

Binding of protein uH2A and histone H2A to DNA

The binding properties of protein uH2A and histone H2A to DNA were investigated by filter binding assays. Both proteins revealed similar affinity for native and denatured DNA. Competition with increasing amounts of repetitive and nonrepetitive DNA has shown that protein uH2A binds selectively to nonrepetitive sequences. When poly d(A-T) was used as a competitor, uH2A bound to this polynucleotide with much greater affinity than histone H2A. These findings suggest a selective binding to regulatory A-T rich intergenic sequences in native DNA.     

Methylation of histone H4 lysine 20 controls recruitment of Crb2 to sites of DNA damage

Histone lysine methylation is a key regulator of gene expression and heterochromatin function, but little is known as to how this modification impinges on other chromatin activities. Here we demonstrate that a previously uncharacterized SET domain protein, Set9, is responsible for H4-K20 methylation in the fission yeast Schizosaccharomyces pombe. Surprisingly, H4-K20 methylation does not have any apparent role in the regulation of gene expression or heterochromatin function. Rather, we find the modification has a role in DNA damage response. Loss of Set9 activity or mutation of H4-K20 markedly impairs cell survival after genotoxic challenge and compromises the ability of cells to maintain checkpoint mediated cell cycle arrest. Genetic experiments link Set9 to Crb2, a homolog of the mammalian checkpoint protein 53BP1, and the enzyme is required for Crb2 localization to sites of DNA damage. These results argue that H4-K20 methylation functions as a "histone mark" required for the recruitment of the checkpoint protein Crb2.     

Accumulation of checkpoint protein 53BP1 at DNA breaks involves its binding to phosphorylated histone H2AX

53BP1 participates in the cellular response to DNA damage. Like many proteins involved in the DNA damage response, 53BP1 becomes hyperphosphorylated after radiation and colocalizes with phosphorylated H2AX in megabase regions surrounding the sites of DNA strand breaks. However, it is not yet clear whether the phosphorylation status of 53BP1 determines its localization or vice versa. In this study we mapped a region upstream of the 53BP1 C terminus that is required and sufficient for the recruitment of 53BP1 to these DNA break areas. In vitro assays revealed that this region binds to phosphorylated but not unphosphorylated H2AX. Moreover, using H2AX-deficient cells reconstituted with wild-type or a phosphorylation-deficient mutant of H2AX, we have shown that phosphorylation of H2AX at serine 140 is critical for efficient 53BP1 foci formation, implying that a direct interaction between 53BP1 and phosphorylated H2AX is required for the accumulation of 53BP1 at DNA break sites. On the other hand, radiation-induced phosphorylation of the 53BP1 N terminus by the ATM (ataxia-telangiectasia mutated) kinase is not essential for 53BP1 foci formation and takes place independently of 53BP1 redistribution. Thus, these two damage-induced events, hyperphosphorylation and relocalization of 53BP1, occur independently in the cell.     

MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks

Histone variant H2AX phosphorylation in response to DNA damage is the major signal for recruitment of DNA-damage-response proteins to regions of damaged chromatin. Loss of H2AX causes radiosensitivity, genome instability, and DNA double-strand-break repair defects, yet the mechanisms underlying these phenotypes remain obscure. Here, we demonstrate that mammalian MDC1/NFBD1 directly binds to phospho-H2AX (gammaH2AX) by specifically interacting with the phosphoepitope at the gammaH2AX carboxyl terminus. Moreover, through a combination of biochemical, cell-biological, and X-ray crystallographic approaches, we reveal the molecular details of the MDC1/NFBD1-gammaH2AX complex. These data provide compelling evidence that the MDC1/NFBD1 BRCT repeat domain is the major mediator of gammaH2AX recognition following DNA damage. We further show that MDC1/NFBD1-gammaH2AX complex formation regulates H2AX phosphorylation and is required for normal radioresistance and efficient accumulation of DNA-damage-response proteins on damaged chromatin. Thus, binding of MDC1/NFBD1 to gammaH2AX plays a central role in the mammalian response to DNA damage.     

Phosphorylation of histone H2AX at M phase in human cells without DNA damage response

A variant of histone H2A, H2AX, is phosphorylated on Ser139 in response to DNA double-strand breaks (DSBs), and clusters of the phosphorylated form of H2AX (gamma-H2AX) in nuclei of DSB-induced cells show foci at breakage sites. Here, we show phosphorylation of H2AX in a cell cycle-dependent manner without any detectable DNA damage response. Western blot and immunocytochemical analyses with the anti-gamma-H2AX antibody revealed that H2AX is phosphorylated at M phase in HeLa cells. In ataxia-telangiectasia cells lacking ATM kinase activity, gamma-H2AX was scarcely detectable in the mitotic chromosomes, suggesting involvement of ATM in M-phase phosphorylation of H2AX. Single-cell gel electrophoresis assay and Western blot analysis with the anti-phospho-p53 (Ser15) antibody indicated that H2AX in human M-phase cells is phosphorylated independently of DSB and DNA damage signaling. Even in the absence of DNA damage, phosphorylation of H2AX in normal cell cycle progression may contribute to maintenance of genomic integrity.     

Critical role of monoubiquitination of histone H2AX protein in histone H2AX phosphorylation and DNA damage response

DNA damage response is an important surveillance mechanism used to maintain the integrity of the human genome in response to genotoxic stress. Histone variant H2AX is a critical sensor that undergoes phosphorylation at serine 139 upon genotoxic stress, which provides a docking site to recruit the mediator of DNA damage checkpoint protein 1 (MDC1) and DNA repair protein complex to sites of DNA breaks for DNA repair. Here, we show that monoubiquitination of H2AX is induced upon DNA double strand breaks and plays a critical role in H2AX Ser-139 phosphorylation (γ-H2AX), in turn facilitating the recruitment of MDC1 to DNA damage foci. Mechanistically, we show that monoubiquitination of H2AX induced by RING finger protein 2 (RNF2) is required for the recruitment of active ataxia telangiectasia mutated to DNA damage foci, thus affecting the formation of γ-H2AX. Importantly, a defect in monoubiquitination of H2AX profoundly enhances ionizing radiation sensitivity. Our study therefore suggests that monoubiquitination of H2AX is an important step for DNA damage response and may have important clinical implications for the treatment of cancers.     

Histone H2AX is phosphorylated at sites of retroviral DNA integration but is dispensable for postintegration repair

The histone variant H2AX is rapidly phosphorylated (denoted gammaH2AX) in large chromatin domains (foci) flanking double strand DNA (dsDNA) breaks that are produced by ionizing radiation or genotoxic agents and during V(D)J recombination. H2AX-deficient cells and mice demonstrate increased sensitivity to dsDNA break damage, indicating an active role for gammaH2AX in DNA repair; however, gammaH2AX formation is not required for V(D)J recombination. The latter finding has suggested a greater dependence on gammaH2AX for anchoring free broken ends versus ends that are held together during programmed breakage-joining reactions. Retroviral DNA integration produces a unique intermediate in which a dsDNA break in host DNA is held together by the intervening viral DNA, and such a reaction provides a useful model to distinguish gammaH2AX functions. We found that integration promotes transient formation of gammaH2AX at retroviral integration sites as detected by both immunocytological and chromatin immunoprecipitation methods. These results provide the first direct evidence for the association of newly integrated viral DNA with a protein species that is an established marker for the onset of a DNA damage response. We also show that H2AX is not required for repair of the retroviral integration intermediate as determined by stable transduction. These observations provide independent support for an anchoring model for the function of gammaH2AX in chromatin repair.     

Binding of small phosphorylated chromatin peptides to DNA

Low-molecular-weight peptides involved in gene expression and cell growth have been isolated from DNA preparation from eukaryotic cells. After phosphorylation with protein kinase CKII (pCKII) these peptides are able to bind to DNA in presence of divalent cations and salt/ethanol. This finding may explain the mechanism by which the peptides exert their activity.     

Structure of C-terminal tandem BRCT repeats of Rtt107 protein reveals critical role in interaction with phosphorylated histone H2A during DNA damage repair

Rtt107 (regulator of Ty1 transposition 107; Esc4) is a DNA repair protein from Saccharomyces cerevisiae that can restore stalled replication forks following DNA damage. There are six BRCT (BRCA1 C-terminal) domains in Rtt107 that act as binding sites for other recruited proteins during DNA repair. Several Rtt107 binding partners have been identified, including Slx4, Rtt101, Rad55, and the Smc5/6 (structural maintenance of chromosome) protein complex. Rtt107 can reportedly be recruited to chromatin in the presence of Rtt101 and Rtt109 upon DNA damage, but the chromatin-binding site of Rtt107 has not been identified. Here, we report our investigation of the interaction between phosphorylated histone H2A (γH2A) and the C-terminal tandem BRCT repeats (BRCT(5)-BRCT(6)) of Rtt107. The crystal structures of BRCT(5)-BRCT(6) alone and in a complex with γH2A reveal the molecular basis of the Rtt107-γH2A interaction. We used in vitro mutagenesis and a fluorescence polarization assay to confirm the location of the Rtt107 motif that is crucial for this interaction. In addition, these assays indicated that this interaction requires the phosphorylation of H2A. An in vivo phenotypic analysis in yeast demonstrated the critical role of BRCT(5)-BRCT(6) and its interaction with γH2A during the DNA damage response. Our results shed new light on the molecular mechanism by which Rtt107 is recruited to chromatin in response to stalled DNA replication forks.     

Threonine 68 of Chk2 is phosphorylated at sites of DNA strand breaks

The protein kinase Chk2 has been implicated in signaling DNA damage to cell cycle checkpoints. In response to ionizing radiation, Chk2 becomes rapidly phosphorylated at threonine 68 by ataxia-telangiectasia mutated (ATM). Here we show that the Thr(68)-phosphorylated form of Chk2 forms distinct nuclear foci in response to ionizing radiation. Only this activated form of Chk2 localizes at sites of DNA strand breaks. The kinase activity of Chk2 and the number of Chk2 foci formed depend on the severity of DNA damage and gradually decline correlating with the predicted value of slowly re-joining double strand breaks. These results suggest that Chk2 is regulated at the sites of DNA strand breaks in response to ionizing radiation.     

Thr 3 phosphorylated histone H3 concentrates at centromeric chromatin at metaphase

Thr 3 was one of the newly characterized phosphorylation sites on histone H3. However, the functional significance of histone H3 Thr 3 phosphorylation during mitosis is unclear. In this study, SDS-PAGE and Western blotting analysis showed that histone H3 Thr 3 was phosphorylated specially during mitosis in MCF-10A and ECV-304 cells. Using indirect immunofluorescence labeling and laser confocal microscopy, we demonstrated that histone H3 Thr 3 phosphorylation occurred from prophase to anaphase and dephosphorylated completely in telophase. Remarkably, Thr 3 phosphorylated histone H3 mostly concentrated at centromeric chromatin at metaphase, which was distinct with Ser 10 phosphorylation aggregated at the telomere, but similar to that characteristic of Thr 11 phosphorylated H3 which is largely restricted to the centromeric chromatin. Using chromatin immunoprecipitation (ChIP) assay, we provided direct evidence that the Thr 3 phosphorylated H3 is associated with centromeric DNA at metaphase. These findings suggested that at metaphase Thr 3 phosphorylated histone H3 may also participate in kinetochore assembly to promote faithful chromosome segregation and serve as another recognition code for kinetochore proteins.     

Radiation damage to histone H2A by the primary aqueous radicals

Histone H2A has been examined for radiation-induced changes in structure and in amino acid composition. The effects of the individual radical intermediates--the hydroxyl radical, solvated electron, and superoxide radical--have been determined by irradiating in dilute aqueous solution under controlled environmental conditions. Amino acid analysis of irradiated histone H2A shows a selective attack on a few residues; only the aromatic residues, phenylalanine and tyrosine, and the heterocyclic residue, histidine, are significantly decreased. A significant increase in aspartic acid is also observed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the hydroxyl radical is the effective radical for promoting changes in protein structure.     

Binding of histone H1 to DNA is described by an allosteric model

Equilibrium binding data were analyzed to characterize the interaction of the linker histone H1 degrees with unmodified T4 phage DNA. Data were cast into the Scatchard-type plot described by McGhee and von Hippel and fit to their eponymous model for nonspecific binding of ligand to DNA. The data were not fit by the simple McGhee-von Hippel model, nor fit satisfactorily by the inclusion of a cooperativity parameter. Instead, the interaction appeared to be well described by Crothers' allosteric model, in which the higher affinity of the protein for one conformational form of the DNA drives an allosteric transition of the DNA to the conformational form with higher affinity (form 2). At 214 mM Na(+), the observed affinity K for an isolated site on unmodified T4 bacteriophage DNA in the form 2 conformation is 4.5 x 10(7) M(-1). The binding constant for an isolated site on DNA in the conformation with lower affinity, form 1, appears to be about 10-fold lower. Binding affinity is dependent on ion concentration: the magnitude of K is about 10-fold higher at 14 mM (5.9 x 10(8) M(-1) for form 2 DNA) than at 214 mM Na(+) concentration.     

Diverse roles for histone H2A modifications in DNA damage response pathways in yeast

There are many types of DNA damage that are repaired by a multiplicity of different repair pathways. All damage and repair occur in the context of chromatin, and histone modifications are involved in many repair processes. We have analyzed the roles of H2A and its modifications in repair by mutagenizing modifiable residues in the N- and C-terminal tails of yeast H2A and by testing strains containing these mutations in multiple DNA repair assays. We show that residues in both tails are important for homologous recombination and nonhomologous end-joining pathways of double-strand break repair, as well as for survival of UV irradiation and oxidative damage. We show that H2A serine 122 is important for repair and/or survival in each of these assays. We also observe a complex pattern of H2A phosphorylation at residues S122, T126, and S129 in response to different damage conditions. We find that overlapping but nonidentical groups of H2A residues in both tails are involved in different pathways of repair. These data suggest the presence of a set of H2A "damage codes" in which distinct patterns of modifications on both tails of H2A may be used to identify specific types of damage or to promote specific repair pathways.     

Endogenous expression of phosphorylated histone H2AX in tumors in relation to DNA double-strand breaks and genomic instability

Microscopically visible gammaH2AX foci signify the presence of DNA double-strand breaks (dsbs) in irradiated cells. However, large foci are also observed in untreated tumour cells, and high numbers reduce the sensitivity for detecting drug or radiation-induced DNA breaks. SW756 cervical carcinoma cells that express about 50 gammaH2AX foci per cell (i.e., equivalent to the number of breaks produced by about 2Gy) showed similar numbers of dsbs as C33A cells that exhibit fewer than three foci per cell. The possibility that differences in numbers of these endogenous foci could be explained by genomic instability perhaps related to misrepair was examined. For 17cell lines selected from the panel of NCI-60 tumor cells previously characterized for karyotypic complexity [A.V. Roschke, G. Tonon, K.S. Gehlhaus, N. McTyre, K.J. Bussey, S. Lababidi, D.A. Scudiero, J.N. Weinstein, I.R. Kirsch, Karyotypic complexity of the NCI-60 drug-screening panel, Cancer Res. 63 (2003) 8634-8647], there was a significant trend (r=0.6) for cell lines with greater numbers of structural or numerical chromosomal rearrangements to show a higher background expression of gammaH2AX. Moreover, cells from this panel with wild-type p53 showed a significantly lower background level of gammaH2AX than cells with mutant p53. To confirm the importance of p53 expression, endogenous and radiation-induced gammaH2AX expression were analyzed using four isogenic SKOV3 cell lines varying in p53 function. Again, higher gammaH2AX expression was found in SKOV3 cell lines expressing mutant p53 compared to wild-type p53. HFL-1 primary lung fibroblasts showed a progressive increase in gammaH2AX as they moved towards senescence, confirming the importance of telomere instability in the development of at least some gammaH2AX foci. Therefore, the explanation for high endogenous levels of gammaH2AX in some tumor cells appears to be multifactorial and may be best described as a consequence of chromatin instability.     

A role for histone H2B during repair of UV-induced DNA damage in Saccharomyces cerevisiae

To investigate the role of the nucleosome during repair of DNA damage in yeast, we screened for histone H2B mutants that were sensitive to UV irradiation. We have isolated a new mutant, htb1-3, that shows preferential sensitivity to UV-C. There is no detectable difference in bulk chromatin structure or in the number of UV-induced cis-syn cyclobutane pyrimidine dimers (CPD) between HTB1 and htb1-3 strains. These results suggest a specific effect of this histone H2B mutation in UV-induced DNA repair processes rather than a global effect on chromatin structure. We analyzed the UV sensitivity of double mutants that contained the htb1-3 mutation and mutations in genes from each of the three epistasis groups of RAD genes. The htb1-3 mutation enhanced UV-induced cell killing in rad1Delta and rad52Delta mutants but not in rad6Delta or rad18Delta mutants, which are defective in postreplicational DNA repair (PRR). When combined with other mutations that affect PRR, the histone mutation increased the UV sensitivity of strains with defects in either the error-prone (rev1Delta) or error-free (rad30Delta) branches of PRR, but did not enhance the UV sensitivity of a strain with a rad5Delta mutation. When combined with a ubc13Delta mutation, which is also epistatic with rad5Delta, the htb1-3 mutation enhanced UV-induced cell killing. These results suggest that histone H2B acts in a novel RAD5-dependent branch of PRR.