Progress 10/01/01 to 09/30/06
Outputs
The most significant findings of this project have been the identification and characterization of three Drosophila genes, in addition to the previously identified mus308, that are specifically hypersensitive to interstrand crosslinking agents and thus identified as components of the DNA repair pathways essential for repair of this unique form of damage. Of the three new genes identified, mus321, mus322, and mus323, two are the Drosophila orthologs of genes previously identified to play roles in DNA repair in other species. We found that the mus322 gene encodes the Snm1 protein, known to be involved in crosslink repair in yeast, but not previously known to function in this pathway in higher eukaryotes. We showed that this protein is in a different repair pathway than the previously identified mus308, with double mutants showing much greater sensitivity than single mutants in either gene. The second of the two newly identified genes, mus323, we identified as one of the
four Drosophila orthologs of the Rad51 protein. It has been known for some time from work in other systems that homologous recombination is involved in interstrand crosslink repair; however, this was the first demonstration that this function was required in this process in Drosophila. Recent work from other systems indicates a likelihood that this gene will act in the same pathway as snm1, and our prediction would be that it would be epistatic to that mutant but synergistic in its effects with mus308. Finally, we determined that mus321 encoded the Drosophila ortholog of the archeal protein Hef. Mus321 includes an amino-terminal domain containing the motifs characteristic of the Superfamily II helicases, consistent with a role in some ATP-dependent step of DNA repair. Our epistasis studies with mus32, mus308 and mus322 have provided insight into the relationships between these three crosslink hypersensitive mutants. The triple mutant exhibits the greatest sensitivity among all single,
double and triple mutant combinations, suggesting that the 3 genes are not part of a single pathway. Our results indicate that mus322 and mus321 are likely to be in one pathway, while mus308 acts in another independent pathway. The Drosophila Mus308 protein has been demonstrated in our lab to be an active DNA polymerase with unusual substrate specificity, and the human ortholog has more recently been demonstrated to catalyze translesion synthesis, consistent with a role in a damage tolerance pathway that provides some degree of recovery from interstrand crosslink damage. The difference in hypersensitivity between mus308 single mutants and the double mutants suggests that two pathways of repair are somewhat distinct. Further work will be necessary to reconcile the nature of these mutants and their epistatic interactions with the sequential or parallel models of crosslink repair pathways. It will also be essential to do further studies of epistatic interactions with other Drosophila
mutants such as the known translesion polymerase rev3 to ascertain that the full complement of crosslink repair proteins are tested.
Impacts
DNA repair is a topic of great importance in biology, since the changes in DNA associated with failure to correctly repair damage are the basis for everything from evolution, for which the raw material is the accumulated changes in DNA sequence; to aging, one aspect of which is the unrepaired damage to the genome that accumulates over a lifetime; to various diseases such as cancer that are associated with unrepaired damage to important regulatory genes. Most people are aware of the need for using sunscreen, but do not understand the relationship between UV light, DNA damage and DNA repair that is the basis for this practice. Understanding this relationship will aid people in making wise choices with respect to their health. The research in this project has added to our understanding of one of the many types of DNA repair that exist in higher eukaryotes; interstrand crosslink repair. One of the genes discovered, mus321, is related to a human gene known as FANCM, which
is associated with one of the several forms of the human disease Fanconi anemia. The identification of this gene in Drosophila opens an additional pathway for scientists interested in understanding the molecular basis of this disease.
Publications
- None in this period. 2006
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Progress 01/01/05 to 12/31/05
Outputs
Our primary goal continues to be the mapping and characterization of a group of mutations that identify genes involved in DNA repair in Drosophila. These genes were identified on the basis of their phenotype; namely, hypersensitivity to DNA damaging chemicals including methyl methane sulfonate, which alkylates DNA and results in double strand breaks, and nitrogen mustard, which can create interstrand crosslinks. During this period, we have completed mapping the fourth (and last) unmapped mutation in the group of mutants specifically sensitive to interstrand crosslinking reagents. We have tentatively identified this gene as the Drosophila ortholog of a gene (RAD51) with a well known function in DNA recombination; this gene is also known as spnA in Drosophila. We are currently carrying out transposon rescue experiments to confirm this identification. We are currently testing epistatic interactions between the full set of crosslink repair genes, which has uncovered
evidence for at least two partially redundant pathways of interstrand crosslink repair. Identifying the molecular nature of the products of all of these genes provides us with information regarding their unique functions in interstrand crosslink repair, and we are proceeding with biochemical studies to further understand their role in this critical process.
Impacts
We are studying the molecular basis of DNA repair using the model organism Drosophila melanogaster, which provides an ideal system for genetic studies of DNA repair, and provides information relevant to understanding DNA repair in humans. Damage to DNA is critical in the development of human disease (particularly cancer) and aging, and understanding the mechanisms by which DNA damage is repaired is important to understanding these processes. One of the repair genes we are studying is the Drosophila homolog of the FANCM gene, involved in the human disease Fanconi anemia.
Publications
- WILSON, M., WIDDICOMBE, J.H., GOHIL, K., BURTIS, K.C., REZNICK, A.Z., CROSS, C.E., EISERICH J.P. (2005) Are Drosophila a useful model for understanding the toxicity of inhaled oxidative pollutants: a review. Inhalation Toxicology 17:765-774.
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Progress 01/01/04 to 12/31/04
Outputs
Our primary goal continues to be the mapping and characterization of a group of mutations that identify genes involved in DNA repair in Drosophila. These genes were identified on the basis of their phenotype; namely, hypersensitivity to DNA damaging chemicals including methyl methane sulfonate, which alkylates DNA and results in double strand breaks, and nitrogen mustard, which can create interstrand crosslinks. During this period, we have focused our efforts most intensely on mapping the fourth (and last) unmapped mutation in the group specifically sensitive to interstrand crosslinking reagents. We are using a combination of approaches to map this gene, including male recombination experiments that take advantage of the dense collection of transposon inserts isolated by the Drosophila Genome Project to use as landmarks in our mapping experiments. As soon as the region is delimited to a reasonable genetic interval (5-10 candidate genes) we will initiate DNA sequencing
and transposon rescue experiments to determine the specific identity of the mutant gene. At present, we have defined the interval containing this mutant to a region including about fifty genes. Identifying this final gene will be critical to our continued effort to understand the molecular basis of interstrand crosslink repair in higher eukaryotes. We will be including this gene in our ongoing study of the epistatic interactions between crosslink repair genes, which has uncovered evidence for two partially redundant pathways of interstrand crosslink repair. Identifying the molecular nature of the products of all of these genes provides us with information regarding their unique functions in interstrand crosslink repair, and we are proceeding with biochemical studies to further understand their role in this critical process.
Impacts
Damage to DNA is critical in the development of human disease (particularly cancer) and aging, and understanding the mechanisms by which DNA damage is repaired is important to understanding these processes. We are studying the molecular basis of DNA repair using the model organism Drosophila melanogaster, which provides an ideal system for genetic studies of DNA repair, and provides information relevant to understanding DNA repair in humans.
Publications
- LAURENCON, A., ORME, C.M., PETERS, H.K., BOULTON, C. L., VLADAR, E.K., LANGLEY, S.A., BAKIS, E. P., HARRIS, D.T., HARRIS, N.J., WAYSON, S.M., HAWLEY, R.S., BURTIS, K.C.. A Large Scale Screen for Mutagen-Sensitive Loci in Drosophila. Genetics 167:217-231 (2004).
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Progress 01/01/03 to 12/31/03
Outputs
During the past year, we have continued to map and characterize a set of newly identified mutations that identify genes involved in DNA repair in Drosophila. These genes were identified on the basis of their phenotype; namely, hypersensitivity to DNA damaging chemicals including methyl methane sulfonate, which alkylates DNA and results in double strand breaks, and nitrogen mustard, which can create interstrand crosslinks. During this period, we have mapped two of these genes to small defined regions of the genome, and have succeeded in getting a new genomics-based approach to creation of deletions with defined endpoints in this region up and running in the lab. This will allow us to define the precise molecular identity of these two genes in the very near future. In addition, we have molecularly identified one of the new mutations. Using a combination of classical and molecular mapping techniques, we have identified one of the two remaining mutants specifically
hypersensitive to crosslinking agents as a second allele of the Drosophila gene mus323. This gene encodes the Drosophila homolog of the yeast snm1 gene, a known crosslink specific mutant in Saccharomyces cerevisiae. The importance of this observation is that finding a second allele of this gene in our screen provides evidence to support the hypothesis that we are near saturation for this phenotype, and that there are likely to be few if any more autosomal genes that can be mutated to give rise to crosslink sensitivity. This observation will be helpful in our continuing research on the epistatic interactions between crosslink repair genes, which has uncovered evidence for two partially redundant pathways of interstrand crosslink repair. The results from this year's work have provided us with the basis for understanding the biological role of these repair genes, and we are proceeding to undertake biochemical studies to further understand their function.
Impacts
Damage to DNA is critical in the development of human disease (particularly cancer) and aging, and understanding the mechanisms by which DNA damage is repaired is important to understanding these processes. We are studying the molecular basis of DNA repair using the model organism Drosophila melanogaster, which provides an ideal system for genetic studies of DNA repair, and provides information relevant to understanding DNA repair in humans.
Publications
- BURTIS KC (2002). Development. Doublesex in the middle. Science 16:297:1135-1136.
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Progress 01/01/02 to 12/31/02
Outputs
During the past year, we have focused our efforts on mapping and characterization of a set of newly identified mutations that identify genes involved in DNA repair in Drosophila. These genes were identified on the basis of their phenotype; namely, hypersensitivity to DNA damaging chemicals including methyl methane sulfonate, which alkylates DNA and results in double strand breaks, and nitrogen mustard, which can create interstrand crosslinks. By screening with both mutagens, we were able to identify mutations in genes involved with several major DNA repair pathways, including nucleotide excision repair, base excision repair, double strand break repair and interstrand crosslink repair. Our major goal is now to determine which of these mutations are in known DNA repair genes, and which identify new genes involved in this process. From among 6,000 second chromosome lines and 6,200 third chromosome lines, we tested 2,585 and 3,690 lines, respectively, for mutagen
sensitivity. This effort resulted in the identification of 18 second chromosomal mutants, 8 of which were new alleles of known genes (mus201(1 allele), mus205 (4 alleles), okra (1 allele), and rad201 (2 alleles)). The remaining 10 mutants were hypersensitive to MMS and defined 9 new mutagen-sensitive genes (mus212-mus220). We also recovered 60 third chromosomal mutants, 44 of which were alleles of known genes (mus301 (8 alleles), mus302 (5 alleles), mus304 (2 alleles), mus305 (24 alleles), mus308 (3 alleles), and mus312 (2 alleles)). The remaining 16 mutants defined 15 new mutagen-sensitive genes (mus313-mus327). Mutants in seven genes were hypersensitive only to MMS (mus313-mus319), mutants in 4 genes were hypersensitive only to nitrogen mustard(mus320-mus323), and mutants in 4 of these genes (5 total mutants) displayed hypersensitivity to both MMS and nitrogen mustard, (mus324-mus327). We have shown that the nitrogen mustard-sensitive gene mus323 gene encodes the Drosophila homolog
of the yeast snm1 gene, a known crosslink specific mutant in Saccharomyces cerevisiae. One additional nitrogen mustard-specific mutation identifies a novel gene not previously known to be involved in interstrand crosslink repair, and we are currently pursuing the molecular characterization of this gene and its product. We are also carrying out further genetic experiments with the crosslink specific mutations we have discovered, and have uncovered evidence for two partially redundant pathways of interstrand crosslink repair. The results from this years work have provided us with an important resource of new DAN repair mutations, and will provide the basis for our investigations in future years of the project.
Impacts
Damage to DNA is critical in the development of human disease (particularly cancer) and aging, and understanding the mechanisms by which DNA damage is repaired is important to understanding these processes. We are studying the molecular basis of DNA repair using the model organism Drosophila melanogaster, which provides an ideal system for genetic studies of DNA repair, and provides information relevant to understanding DNA repair in humans.
Publications
- BURGERS PM, KOONIN EV, BRUFORD E, BLANCO L, BURTIS KC, CHRISTMAN MF, COPELAND WC, FRIEDBERG EC, HANAOKA F, HINKLE DC, LAWRENCE CW, NAKANISHI M, OHMORI H, PRAKASH L, PRAKASH S, REYNAUD CA, SUGINO A, TODO T, WANG Z, WEILL JC, WOODGATE R. Eukaryotic DNA polymerases: proposal for a revised nomenclature. J Biol Chem. 276:43487-43490 (2001).
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