Recruitment Of Repair Proteins Research Articles

Mrg15 null and heterozygous mouse embryonic fibroblasts exhibit DNA-repair defects post exposure to gamma ionizing radiation

MORF4-related gene on chromosome 15 (MRG15) is a core component of the NuA4/Tip60 histone acetyltransferase complex that modifies chromatin structure. We here demonstrate that Mrg15 null and heterozygous mouse embryonic fibroblasts exhibit an impaired DNA-damage response post gamma irradiation, when compared to wild-type cells. Defects in DNA-repair and cell growth, and delayed recruitment of repair proteins to sites of damage were observed. Formation of phosphorylated H2AX and 53BP1 foci was delayed in Mrg15 mutant versus wild-type cells following irradiation. These data implicate a novel role for MRG15 in DNA-damage repair in mammalian cells.

Novel Role of mfd : Effects on Stationary-Phase Mutagenesis in Bacillus subtilis

Previously, using a chromosomal reversion assay system, we established that an adaptive mutagenic process occurs in nongrowing Bacillus subtilis cells under stress, and we demonstrated that multiple mechanisms are involved in generating these mutations (41, 43). In an attempt to delineate how these mutations are generated, we began an investigation into whether or not transcription and transcription-associated proteins influence adaptive mutagenesis. In B. subtilis, the Mfd protein (transcription repair coupling factor) facilitates removal of RNA polymerase stalled at transcriptional blockages and recruitment of repair proteins to DNA lesions on the transcribed strand. Here we demonstrate that the loss of Mfd has a depressive effect on stationary-phase mutagenesis. An association between Mfd mutagenesis and aspects of transcription is discussed.

Histone H3 and H4 Ubiquitylation by the CUL4-DDB-ROC1 Ubiquitin Ligase Facilitates Cellular Response to DNA Damage

Posttranslational histone modifications play important roles in transcription and other chromatin-based processes. Compared to acetylation, methylation, and phosphorylation, very little is known about the function of histone ubiquitylation. Here, we report the purification and functional characterization of a histone H3 and H4 ubiquitin ligase complex, CUL4-DDB-ROC1. We demonstrate that CUL4-DDB-ROC1-mediated H3 and H4 ubiquitylation occurs both in vitro and in vivo. Importantly, CUL4-DDB-ROC1-mediated H3 and H4 ubiquitylation is regulated by UV irradiation. Reduction of histone H3 and H4 ubiquitylation by knockdown of CUL4A impairs recruitment of the repair protein XPC to the damaged foci and inhibits the repair process. Biochemical studies indicate that CUL4-DDB-ROC1-mediated histone ubiquitylation weakens the interaction between histones and DNA and facilitates the recruitment of repair proteins to damaged DNA. Thus, our studies uncover CUL4-DDB-ROC1 as a histone ubiquitin ligase and demonstrate that histone H3 and H4 ubiquitylation participates in the cellular response to DNA damage.

In UV-irradiated Saccharomyces cerevisiae, overexpression of Swi2/Snf2 family member Rad26 increases transcription-coupled repair and repair of the non-transcribed strand.

Nucleotide excision repair (NER) in eukaryotes is a pathway conserved from yeast to humans that removes many bulky chemical adducts and UV-induced photoproducts from DNA in a relatively error-free manner. In addition to the recognition and excision of DNA damage throughout the genome (GGR), there exists a mechanism, transcription-coupled nucleotide excision repair (TCR), for recognizing some types of DNA damage in the transcribed strand of genes in Escherichia coli, yeast and mammalian cells. An obstacle in the repair of the transcribed strand of active genes is the RNA polymerase complex stalled at sites of DNA damage. The stalled RNA polymerase complex may then mediate recruitment of repair proteins to damage in the transcribed strand. Proteins enabling TCR are the Cockayne syndrome B (CSB) protein in humans and its yeast homologue Rad26. Both CSB and Rad26 belong to the Swi2/Snf2 family of DNA-dependent ATPases, which change DNA accessibility to proteins by altering chromatin structure. To address how Rad26 functions in yeast repair, we used the genetic approach of overexpressing Rad26 and examined phenotypic changes, i.e. changes in NER. We found that repair of both the transcribed and the non-transcribed strands is increased. In addition, overexpression of Rad26 partially bypasses the requirement for Rad7 in GGR, specifically in the repair of non-transcribed sequences. As TCR takes place in very localized regions of DNA (i.e. within genes) in wild-type cells, we propose that overexpression of recombinant Rad26 increases accessibility of the damaged DNA in chromatin for interaction with repair proteins.

Gamma-H2AX expression pattern in non-irradiated neonatal mouse germ cells and after low-dose gamma-radiation: relationships between chromatid breaks and DNA double-strand breaks.

The DNA double-strand breaks (DSBs) are considered to be the most relevant lesions for the deleterious effects of ionizing radiation exposure. The discovery that the induction of DSBs is rapidly followed by the phosphorylation of H2AX histone at Ser-139, favoring repair protein recruitment or access, opens the possibility for a wide range of research. This phosphorylated histone, named gamma-H2AX, has been shown to form foci in interphase nuclei as well as megabase chromatin domains surrounding the DNA lesion on chromosomes. Using detection of gamma-H2AX on germ cell mitotic chromosomes 2 h after gamma-irradiation, we studied radiation-induced DSBs during the G(2)/M phase of the cell cycle. We show that 1) non-irradiated neonatal germ cells express gamma-H2AX with variable patterns at metaphase, 2) gamma-irradiation induces foci whose number increases in a dose-dependent manner, 3) some foci correspond to visible chromatid breaks or exchanges, 4) sticky chromosomes characterizing cell radiation exposure during mitosis are a consequence of DSBs, and 5) gamma-H2AX remains localized at the sites of the lesions even after end-joining has taken place. This suggests that completion of DSB repair does not necessarily imply disappearance of gamma-H2AX.

Relationship of DNA double-strand breaks to synapsis in Drosophila.

The relationship between synaptonemal complex formation (synapsis) and double-strand break formation (recombination initiation) differs between organisms. Although double-strand break creation is required for normal synapsis in Saccharomyces cerevisiae and the mouse, it is not necessary for synapsis in Drosophila and Caenorhabditis elegans. To investigate the timing of and requirements for double-strand break formation during Drosophila meiosis, we used an antibody that recognizes a histone modification at double-strand break sites, phosphorylation of HIS2AV (gamma-HIS2AV). Our results support the hypothesis that double-strand break formation occurs after synapsis. Interestingly, we detected a low (10-25% of wildtype) number of gamma-HIS2AV foci in c(3)G mutants, which fail to assemble synaptonemal complex, suggesting that there may be both synaptonemal complex-dependent and synaptonemal complex-independent mechanisms for generating double-strand breaks. Furthermore, mutations in Drosophila Rad54 (okr) and Rad51 (spnB) homologs cause delayed and prolonged gamma-HIS2AV staining, suggesting that double-strand break repair is delayed but not eliminated in these mutants. There may also be an interaction between the recruitment of repair proteins and phosphorylation.

Insights into ssDNA recognition by the OB fold from a structural and thermodynamic study of Sulfolobus SSB protein.

Information processing pathways such as DNA replication are conserved in eukaryotes and archaea and are significantly different from those found in bacteria. Single-stranded DNA-binding (SSB) proteins (or replication protein A, RPA, in eukaryotes) play a central role in many of these pathways. However, whilst euryarchaea have a eukaryotic-type RPA homologue, crenarchaeal SSB proteins appear much more similar to the bacterial proteins, with a single OB fold for DNA binding and a flexible C-terminal tail that is implicated in protein-protein interactions. We have determined the crystal structure of the SSB protein from the crenarchaeote Sulfolobus solfataricus to 1.26 A. The structure shows a striking and unexpected similarity to the DNA-binding domains of human RPA, providing confirmation of the close relationship between archaea and eukaryotes. The high resolution of the structure, together with thermodynamic and mutational studies of DNA binding, allow us to propose a molecular basis for DNA binding and define the features required for eukaryotic and archaeal OB folds.

MDC1 is a mediator of the mammalian DNA damage checkpoint.

To counteract the continuous exposure of cells to agents that damage DNA, cells have evolved complex regulatory networks called checkpoints to sense DNA damage and coordinate DNA replication, cell-cycle arrest and DNA repair. It has recently been shown that the histone H2A variant H2AX specifically controls the recruitment of DNA repair proteins to the sites of DNA damage. Here we identify a novel BRCA1 carboxy-terminal (BRCT) and forkhead-associated (FHA) domain-containing protein, MDC1 (mediator of DNA damage checkpoint protein 1), which works with H2AX to promote recruitment of repair proteins to the sites of DNA breaks and which, in addition, controls damage-induced cell-cycle arrest checkpoints. MDC1 forms foci that co-localize extensively with gamma-H2AX foci within minutes after exposure to ionizing radiation. H2AX is required for MDC1 foci formation, and MDC1 forms complexes with phosphorylated H2AX. Furthermore, this interaction is phosphorylation dependent as peptides containing the phosphorylated site on H2AX bind MDC1 in a phosphorylation-dependent manner. We have shown by using small interfering RNA (siRNA) that cells lacking MDC1 are sensitive to ionizing radiation, and that MDC1 controls the formation of damage-induced 53BP1, BRCA1 and MRN foci, in part by promoting efficient H2AX phosphorylation. In addition, cells lacking MDC1 also fail to activate the intra-S phase and G2/M phase cell-cycle checkpoints properly after exposure to ionizing radiation, which was associated with an inability to regulate Chk1 properly. These results highlight a crucial role for MDC1 in mediating transduction of the DNA damage signal.

Human alpha spectrin II and the FANCA, FANCC, and FANCG proteins bind to DNA containing psoralen interstrand cross-links.

Repair of DNA interstrand cross-links is a complex process critical to which is the identification of sites of damage by specific proteins. We have recently identified the structural protein nonerythroid alpha spectrin (alphaSpIISigma) as a component of a nuclear protein complex in normal human cells which is involved in the repair of DNA interstrand cross-links and have shown that it forms a complex with the Fanconi anemia proteins FANCA, FANCC, and FANCG. Using DNA affinity chromatography, we now show that alphaSpIISigma, present in HeLa cell nuclei, specifically binds to DNA containing psoralen interstrand cross-links and that the FANCA, FANCC, and FANCG proteins are bound to this damaged DNA as well. That spectrin binds directly to the cross-linked DNA has been shown using purified bovine brain spectrin (alphaSpIISigma1/betaSpIISigma1)2. Binding of the Fanconi anemia (FA) proteins to the damaged DNA may be either direct or indirect via their association with alphaSpIISigma. These results demonstrate a role for alpha spectrin in the nucleus as well as a new function for this protein in the cell, an involvement in DNA repair. alphaSpIISigma may bind to cross-linked DNA and act as a scaffold to help in the recruitment of repair proteins to the site of damage and aid in their alignment and interaction with each other, thus enhancing the efficiency of the repair process.

Expansions and contractions in 36-bp minisatellites by gene conversion in yeast.

The instability of simple tandem repeats, such as human minisatellite loci, has been suggested to arise by gene conversions. In Saccharomyces cerevisiae, a double-strand break (DSB) was created by the HO endonuclease so that DNA polymerases associated with gap repair must traverse an artificial minisatellite of perfect 36-bp repeats or a yeast Y' minisatellite containing diverged 36-bp repeats. Gene conversions are frequently accompanied by changes in repeat number when the template contains perfect repeats. When the ends of the DSB have nonhomologous tails of 47 and 70 nucleotides that must be removed before repair DNA synthesis can begin, 16% of gene conversions had rearrangements, most of which were contractions, almost always in the recipient locus. When efficient removal of nonhomologous tails was prevented in rad1 and msh2 strains, repair was reduced 10-fold, but among survivors there was a 10-fold reduction in contractions. Half the remaining events were expansions. A similar decrease in the contraction rate was observed when the template was modified so that DSB ends were homologous to the template; and here, too, half of the remaining rearrangements were expansions. In this case, efficient repair does not require RAD1 and MSH2, consistent with our previous observations. In addition, without nonhomologous DSB ends, msh2 and rad1 mutations did not affect the frequency or the distribution of rearrangements. We conclude that the presence of nonhomologous ends alters the mechanism of DSB repair, likely through early recruitment of repair proteins including Msh2p and Rad1p, resulting in more frequent contractions of repeated sequences.

Poly(ADP-ribose) polymerase, a major determinant of early cell response to ionizing radiation

Purpose : To determine whether DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase (PARP-1) are involved in eliciting the rapid fluctuations of radiosensitivity that have been observed when cells are exposed to short pulses of ionizing radiation. Materials and methods : The effect of DNA-PK and PARP-1 inhibitors on the survival of cells to split-dose irradiation was investigated using Chinese hamster V79 fibroblasts and human carcinoma SQ-20B cells. The responses of PARP-1 proficient and PARP-1 knockout mouse 3T3 fibroblasts were compared in a similar split-dose assay. Results : Inactivation of DNA-PK by wortmannin potentiated radiation-induced cell kill but it did not alter the oscillatory, W-shaped pattern of early radiation response. In contrast, oscillatory radiation response was abolished by 3-aminobenzamide, a reversible inhibitor of enzymes containing a PARP catalytic domain. The oscillatory response was also lacking in PARP-1 knockout mouse 3T3 fibroblasts. Conclusion : The results show that PARP-1 plays a key role in the earliest steps of cell response to ionizing radiation with clonogenic ability or growth as endpoint. It is hypothesized that rapid poly(ADP-ribosylation) of target proteins, or recruitment of repair proteins by activated PARP-1 at the sites of DNA damage, bring about rapid chromatin remodelling that may affect the incidence of chromosomal damage upon re-irradiation.