Characterization of O6-Methylguanine DNA Methyltransferase and Base Excision DNA Repair Activity in Xiphophorous Fishes

Treatment of cells and/or DNA with direct acting alkylating agents (i.e. methyl-methane sulfonate; MMS, methyl nitrosourea; MNU, N-methyl-N-nitro-N nitrosoguanidine; MNNG) produces a wide spectrum of DNA adducts. The most frequent base adducts formed by alkylating agent exposure include those produced by alkylation of purine nucleophilic centers (N7 and N3 alkylpurines). Alkylation at the 06 position of guanine and o4 position of thymine occurs at much lower frequencies. Alkylpurine lesions (N7 and N3) are primarily responsible for cytotoxicity associated with alkylation treatment and are repaired principally by base excision repair (BER) mechanisms. In contrast, 09-alkylguanine and O4-alkylthymine lesions are believed primarily responsible for the mutagenic (and thus carcinogenic) effects of DNA alkylation, but do not contribute appreciably to cytotoxicity. The O9-alkyl guanine lesion is repaired by a highly conserved DNA repair protein, O9-methyl guanine DNA methyltransferase (06-MGMT). Xiphophorus fish provide an excellent system to study the heritable factors of tumorigenesis and carcinogenesis since inter-species cross are fertile and several select inter-species crosses produce progeny which show enhanced susceptibility to MNU induced tumor formation. Preliminary studies in our laboratory have indicated that Xiphophorus fish parental stocks utilized in tumor induction crosses may possess differential toxicity to the alkylating agents MNU and MNNG. It seems reasonable to hypothesize that such differences in toxicity and tumor susceptibility may be dependent on the capabilities of each stock to perform DNA repair after alkylating agent treatment. Since very little is known about DNA repair in lower vertebrates, studies were undertaken to first investigate O6-MGMT activity in cellular extracts derived from several Xiphophorus fish species used in tumor susceptibility experiments. To perform these studies a custom synthesized double stranded oligonucleotide (18 mer) containing a single O6-MG residue was utilized to characterize X Xiphophorus O6-MGMT and to determine the relative O6-MGMT activity in cellular extracts derived from brain, gill and liver tissues harvested from various Xiphophorus fishes. Also presented are results of experiments aimed at determination of BER capacity in inbred Xiphophorus parental stocks and inter-species F1 hybrid animals. These BER Studies were performed using a double stranded oligonucleotide specifically designed to allow measurement of cytosine incorporation at a G:U mismatch by BER repair enzymes in crude tissue extracts. Overall the studies reported herein provide basic data that lead to a better understanding of DNA repair in intact vertebrate animals and this information will be useful to the design of future studies aimed at understanding the relationships between DNA repair, mutagenesis, and latent tumorigenesis in this valuable fish model system.

Name thesis:degree_name

Master of Science

Level thesis:degree_level

Masters

Discipline thesis:degree_discipline

Biology

Grantor

Southwest Texas State University

Year dc:date.issued

1997

Author dc:creator

• Sung, Haung-Mo

Advisor dc:contributor.advisor

• Walter, Ronald B.

Committee members dc:contributor.committeemember

• Horne, Francis Ray
• McLean, Robert J. C.

• DNA repair
• Xiphophorus
• alkylating agents

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/10877/21843

OAI identifier oai:identifier

oai:digital.library.txst.edu:10877/21843