Alkylating agents represent an abundant class of chemical DNA damaging agents in our environment and they are toxic, mutagenic, teratogenic and carcinogenic. Since we are continuously exposed to alkylating agents, and since certain alkylating agents are used for cancer chemotherapy, it is important to understand how cells respond when exposed to these agents. The repair of DNA alkylation damage provides tremendous protection from the toxic effects of these agents and our aim is to understand the biology, the biochemistry, and the genetics of numerous DNA repair pathways that act upon DNA alkylation damage.
Toxicogenomics is a new scientific field that elucidates how the entire genome is involved in biological responses of organisms exposed to environmental toxicants/stressors. Our laboratory uses transcriptional profiling to study gene expression changes induced by DNA damaging chemicals in order to better understand their mechanisms of action. We perform studies in a variety of model systems including yeast, bacteria, cultured mammalian cells, and in vivo experiments in mice. We are fully equipped to run Affymetrix GeneChip© oligonucleotide arrays or a variety of other platforms through our collaborations with MIT BioMicroCenter
We are a member of the Toxicogenomics Research Consortium whch consists of six Cooperative Research Members (CRMs), two Resource Contractors, and NIEHS Extramural Staff. The CRMs include 5 academic centers and the NIEHS Microarray Group. These include: Duke University, Fred Hutchinson Cancer Research Center, MIT, NIEHS Microarray Group, OHSU
, and UNC.
Animal Models of DNA Repair
Humans are exposed to alkylating agents from the environment, and DNA-alkylation intermediates are constantly formed during normal cellular metabolism. Furthermore, certain alkylating agents are used for cancer chemotherapy and pose danger to healthy tissues. Thus, it is important to understand how cells in vivo are affected by these agents. One fruitful approach is to study mouse models that are deficient in specific DNA repair genes, or that overproduce repair enzymes. Our laboratory has developed transgenic and knock-out mice with altered DNA repair capabilities and we are studying their susceptibility to alkylating agent-induced toxicity and carcinogenesis.
DNA Repair Pathways
We are studying the biology of E. coli AlkB and its human relatives, hABH1, hABH2 and hABH3. AlkB has long been known to play a role in protecting cells from alkylation damage, and only recently has it been shown to repair damaged cytosine and adenine in DNA through a novel mechanism, oxidative demethylation. Surprisingly, AlkB and hABH3 can even repair RNA.
We are also studying DNA repair pathways dealing with alkylated base lesions present in cellular DNA. (i) Base excision repair (BER), initiated when the damaged base is enzymatically removed by a 3-methyladenine (3MeA) DNA glycosylase. (ii) Direct reversal of base damage wherein the unwanted alkyl group on O6-methylguanine (O6-MeG) is transferred to a DNA repair methyltransferase (MTase), thus inactivating the MTase and restoring the normal DNA base.
We are examining the global transcriptional response of mammalian cells and tissues to carcinogenic alkylating agents, and in addition to examine the details of this global picture that represent the response elicited by the presence of specific alkylated bases in the genome (e.g., 3MeA and O6MeG). For this, we exploit two knock-out mouse model systems that we recently generated. One is deficient in 3MeA DNA glycosylase repair activity (Aag null mice), and the other is deficient in the O6-MeG DNA repair MTase activity (Mgmt null mice).
We have generated a genomic phenotyping database identifying hundreds of S. cerevisiae genes important for viable cellular recovery after mutagen exposure. Systematic phenotyping of 1,615 gene deletion strains produced distinctive signatures for each of four mutagens. Computational integration of the database with 4,025 interacting proteins, comprising the yeast interactome, identified several multi-protein networks important for damage recovery. Some networks were associated with DNA metabolism and cell cycle control functions, but most were associated with unexpected functions such as cytoskeleton remodeling, chromatin remodeling, and protein, RNA and lipid metabolism. Hence, a plethora of responses other than the DNA damage response are important for recovery.
An increase in spontaneous mutations is associated with increased cancer risk. Our laboratory has shown that a simple imbalance between the first two enzymes involved in DNA base excision repair can increase
the rate of spontaneous mutations several hundred-fold. Specifically, the S. cerevisiae MAG1 3-methyl-adenine DNA glycosylase, when expressed at high levels relative to the apurinic/apyrimidinic endonuclease (APN1), increases spontaneous mutation by up to approximately 600-fold in S. cerevisiae and approximately 200-fold in E. coli.
Genetic evidence suggests that, in yeast, the increased spontaneous mutation requires the generation of abasic sites and the processing of these sites by the REV1/REV3/REV7 lesion bypass pathway.
Gene Environment Interactions
When cells are exposed to DNA damaging agents a signal is generated such that the transcription of various genes is altered. We have used Affymetrix oligonucleotide DNA chip analysis to monitor the transcriptional response of the entire S. cerevisiae genome (i.e., all 6,200 genes) in response to a number of different alkylating agents. To our surprise, we have identified hundreds of responsive genes and have uncovered a hitherto unknown response that links ubiquitin-mediated protein degradation and DNA repair. We are currently exploring the biological roles that the large number of responsive genes plays in protecting cells against alkylation toxicity. Signals can also be generated, in cells exposed to alkylating agents, which trigger cell cycle checkpoint arrest or apoptosis. We are also dissecting the molecular mechanisms by which alkylating agents signal these very important downstream events.
Structure Function Relationship
The human 3-methyladenine DNA glycosylase (AAG) catalyzes the first step of base excision repair by cleaving damaged bases from DNA. The structure of AAG complexed to DNA suggests how modified bases can be distinguished from normal DNA bases in the enzyme active site. Mutational analyses of residues contacting the alkylated base suggest that the shape of the damaged base, its hydrogen-bonding characteristics, and its aromaticity all contribute to the selective recognition of damage by AAG.