Double-strand Breaks And Untargeted Dna Metabolic Events

Resnick, Michael

Abstract

Chromatin functions in repair and recombination. Assembly of new chromatin during S phase requires multiple histone chaperone complexes, including CAF-1 (Cac2p, Msi1p and Rlf2p) and RCAF (Asf1p plus acetylated forms of histones H3 and H4) in the yeast Saccharomyces cerevisiae. Cells lacking both CAF-1 and RCAF are viable, but are hypersensitive to several chemical DNA damaging agents known to cause breaks in chromosomal DNA. We have addressed the role of these complexes in the repair of DSBs by the homologous recombination and nonhomologous end-joining (NHEJ) pathways. Specifically we analyzed the ability of yeast cells lacking CAF-1, RCAF, or both complexes to carry out repair of a single DSB in substrates that report recombination between a transformed plasmid and chromosome or endjoining in a transformed plasmid. We established that cells lacking replication-dependent chromatin assembly factors were defective in repair of site-specific DSBs by both major pathways of repair (recombination and NHEJ). We then determined that the reduction in NHEJ repair was an indirect effect caused by derepression of silenced mating loci at HML and HMR and consequent production of the a1/alpha2 repressor complex. Recently we determined if the impact of chromatin assembly factors was specific to recombination events involving the highly condensed chromosomal DNA normally found in the nucleus or would also be seen in assays using circular plasmids. The DNA of small plasmids (also called mini-chromosomes) contains nucleosome complexes, but lacks many of the proteins and structural features of chromosomes. Defects in chromosome recombination seen in cells lacking CAF were also observed when the recombination target was an extrachromosomal plasmid. We interpreted these results to be an indication that the observed recombination defects are unlikely to involve access of repair enzymes to the DNA. Rather, we propose that replication-dependent reassembly of nucleosomes is an important step during normal recombinational repair of DSBs, possibly coupled to the new DNA synthesis that occurs during strand exchange. Identification of genes required for resistance to double-strand breaking agents. The cellular response to DNA damaging agents involves many genes and pathways. We have taken a systematic approach to identifying all genes that impact on survival/growth response to ionizing radiation. We have now completed the genome wide screening of the total diploid deletion strain collection, for sensitivity to a single dose (80 krads) of ionizing radiation (IR) as previously described (Bennett et al., 2001). The strain collection, which was developed by the Yeast Deletion Project, provides deletions of all nonessential genes or ORFs. Our recent work completes and extends our previous study in that the ~1200 gene deletions remaining to be screened for radiation sensitivity were also screened for sensitivity to other DNA damaging agents including ultraviolet light (UV), methyl methane sulphonate (MMS), hydroxyurea (HU), bleomycin, camptothecin, and doxorubicin. A total of 4746 strains with deletions in each nonessential gene and ORF have been examined. We identified 199 genes that confer IR resistance with many affecting replication, recombination and checkpoint functions. Only 31 were previously identified as having roles in repair or checkpoint functions; therefore, 165 were newly identified as affecting IR sensitivity. Thus ~4% of the nonessential genes are required for toleration of IR damage. With the completion of this screen, we have determined for the first time the total complement of nonessential genes required for the toleration of IR damage. Approximately 90% of the genes affect resistance to other DNA damaging agents including bleomycin, doxorubicin, methyl methanesulfonate (MMS), hydroxyurea (HU), camptothecin and ultraviolet light (UV). Based on this cross sensitivity, new and previously identified radiation sensitive mutants (i.e., RAD52, RAD9, etc.) could be placed into at least 22 different groups. Based on this grouping and comparing relative sensitivities of haploid as well as diploid strains to IR, MMS, HU and UV, one ORF--YLR320W--has now been designated MMS22. Preliminary characterization of this gene suggests that it may be involved in the S phase checkpoint. Among the 165 new loci, 119 correspond to genes for which a function or genetic role has been suggested or experimentally determined. Most of these genes could be placed into 9 functional groups containing at least 5 members. Three ORFs were placed into functional groups based on predicted protein function from sequence homology with previously characterized genes. The protein products comprising these 9 functional groups are associated with chromatin remodeling, silencing or telomere function; stable chromosome transmission during mitosis; structural elements of the nuclear pore complex; transcription and/or mRNA stability; golgi/vacuolar activities; ubiquitin-mediated protein degradation; components of the cytoskeleton/spindle apparatus required for cytokinesis; mitochondrial function; components of the cell wall and/or heat shock stress responses. The genes in these functional groups had not been previously described as affecting responses to IR. Representatives of some groups may have multiple functions and can also be placed in other functional groups. Therefore, this approach has identified new processes and/or targets that can affect responses to radiation. Epistasis analysis is useful in determining if two radiation resistance genes reside in the same repair pathway. Genes are epistatic (in the same pathway) if one mutant gene masks the radiation sensitivity of a second mutant. Haploid and diploid double deletion strains that contained a rad6, 52, 50 or rad9 deletion were constructed for a small number of mutations. These mutants were used to identify CCR4 as a member of the RAD9 epistasis pathway. The ability to obtain viable haploid or diploid double deletions of asf1, mms22 and lip5 with either rad9 or rad52 indicates that when these genes are simultaneously inactivated they are not synthetically lethal. The inability to obtain cnm67 or vid31 with rad9 or rad52 suggests either there are gene dosage effects in meiosis when combining these alleles or alternatively, genetic rearrangements that inhibit meiosis may be present in the cnm67_ and vid31_ haploid strains. However, the ability to obtain dhh1_rad9_ double mutants but not dhh1_rad52_ or dhh1_rad6_ double mutants suggests that a DHH1 may be synthetically lethal when combined with RAD6 or RAD52 mutants. These studies provide new opportunities to understand the totality of response to environmental agents, in this case ionizing radiation. Mechanisms of radiation toleration have largely been considered to be in processes related to chromosome metabolism and DSB repair. Defective genes that result in severe radiation sensitivity in yeast are typically in pathways that directly affect recombinational repair or checkpoint function. However, processes that relate to chromosomal metabolism can be anticipated to be involved in radiation resistance as well. IR resistance genes are proposed to also affect indirectly recombinational and checkpoint proteins by influencing the protein stability of gene products that participate in these processes. We have further suggested that some of the IR resistance genes may affect organelle function such that these essential targets become sensitized. Also, some gene mutations may result in sensitivity to lesions other than DNA DSBs. Many new genes were identified that are transcriptional regulators suggesting that damage-inducible mechanisms play a more important role in resistance to radiation damage than previously thought. Some genes may define entirely new pathways of IR damage