DNA repair focus of Bobola Lab

O6-methylguanine-DNA methyltransferase. O6-methylguanine-DNA methyltransferase (MGMT) is a 22,000 Dalton protein that specifically removes small alkyl groups from the O6 position of guanine onto itself. The alkyl receptor site on MGMT is not regenerated, limiting the number of O6-alkyl-guanine adducts that can be removed in vivo to the number of MGMT molecules and the rate of synthesis of new MGMT. In vitro studies have demonstrated the effectiveness of MGMT in protecting cells from the cytotoxic effects of chemotherapeutic mono- and bi- functional alkylating drugs. A direct biochemical relationship between MGMT activity and resistance has been demonstrated using MGMT inhibitors. In addition to MGMT’s relationship to resistance, MGMT activity is associated with tumorigenesis and recurrence in response to alkylating agent-based therapies.

An appreciable fraction of alkylating agent resistance in pediatric brain tumor cell lines is independent of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT).

Base excision repair. Base excision repair (BER) is a ubiquitous defense against DNA damage that has been conserved from bacteria to man. It is responsible for repairing numerous altered bases; e.g. those produced by alkylation, oxidation, and spontaneous deamination. The alterations recognized and repaired by BER generally do not cause large distortions in the DNA helix. BER is initiated by the hydrolysis of the N-glycosylic linkage between the base and the deoxyribose producing an abasic site. Hydrolysis of the N-glycosylic linkage can occur spontaneous or the action of DNA glycosylases. The resulting abasic site is repaired by one of two pathways. In the major pathway, a Mg ++ dependent apurinic/apyrimidinic endonuclease (APE) nicks 5’to the abasic site, leaving a 5’-deoxyribose-phosphate, Which is excised by the 5’-deoxyribose phophodiesterase activity of the 8 kDa Nterminal domain of DNA polymerase b. The resulting gap is then filled in by DNA polymerase b and ligated by DNA ligase III.

Alkyladenine DNA Glycosylases. Alkyladenine DNA glycosylases (AAG), also known as 3-methyladenine- DNA glycosylase is a 32 000 Dalton protein that excises alkylated bases from DNA yielding abasic sites. Human AAG displays activity against a diverse range of alkylated nucleosides; N3-meA, N3-meG and N7-meG ethylated A and G and 1,N6-etheno-A, 3,N4etheno-C N2,3-etheno-G and 1N2-etheno-G. The action of AAG produces an abasic site, the repair of which is predominantly by apurinic/apyrimidinic endonuclease (APE).

Apurinic/Apyrimidinic Endonuclease. Abasic sites in DNA, generated either by spontaneous depurination/depyrimidination or the action of DNA glycosylases, are the most common lesions in cells. In addition, alkylation at the N3 or N7 atoms of purines or the O2 atom of pyrimidines increases the instability of the N-glycosylic bond in deoxyribonucleosides by orders of magnitude. The lethality of abasic sites is manifested in the alkylating agent hypersensitivity of both prokaryotic and eukaryotic mutants deficient in APE activity. The major human apurinic/apyrimidinic endonuclease (APE) is a 37 000 Dalton protein that repairs the majority of abasic sites. Besides its repair activity APE regulates the DNA-binding capability of a number of transcription factors via a reduction/oxidation (redox) mechanism. The DNA repair and redox activities are localized to two distinct domains within the protein. The DNA repair domain, cterminal, of human APE, is structurally and functionally related to the major APEs of E.coli Saccharomyces cerevisiae, Drosophila and other mammals. The n-terminal domain, necessary for the redox activity, is unique to mammals. The DNA repair activity of APE cleaves the phosphodiester bond 5’ to abasic site yielding 5’-phosphoryl and 3’-hydroxyl ends. APE mutants have been developed that have near wild type binding affinity, to Abasic sites, while displaying a wide range in cleavage rates demonstrating that the substrate binding and cleavage are genetically separable. The major yeast APE protect S. cerevisiae from the toxic effects of alkylating agents and the E. coli enzymes, exonuclease III and Endonuclease IV, protect E. coli against toxicity to ionizing radiation and alkylating agents. Expression of the yeast APE in repair deficient E. coli confer resistance to a number of DNA alkylating agents and expression of the human APE in repair deficient E. coli confer resistance to MMS and ionizing radiation. In addition to its DNA repair activity, APE also regulates the DNA-binding activity of a number of transcription factors, e.g. NF kappa b, Myb, p53, members of the ATF/CREB family and the AP-1 binding proteins Fos and Jun. APE appears to be necessary in early embryonic development. Homozygous mutant mice, lacking a functional APE gene, die during early embryonic development. In contrast, heterozygous mice develop into adulthood without any apparent abnormalities. APE is up-regulated in response to reactive oxygen species which not only increases its DNA repair activity but also its effect on transcription factors. These findings suggest that APE is a major factor in both development and in cellular response to stress/ DNA damage. APE and pol b are up-regulated in response to reactive oxygen. APE and DNA pol b work in concert to repair DNA, APE produces single nucleotide gaps in the DNA which are repaired predominantly by DNA pol b, APE’s close association with pol b activity makes APE activity an important factor to consider when evaluating pol b’s relationship with tumor response to therapy.

DNA polymerase. Mammalian DNA Polymerase b has long been considered a primary catalyst for DNA repair synthesis, particularly short patch repair. DNA Pol b is most likely responsible for DNA repair synthesis following removal of abasic sites by APE and for repair synthesis following recognition and removal of a number of other DNA adducts including bulky chemical adducts, protein-DNA cross-links and intra-stand cross-links, caused by chemo and radiation therapy. In vitro studies show that the rate-limiting step in base excision repair is the removal of deoxyriboncleotide phosphate (dRP) i.e. dRP lyase, catalyzed by the amino-terminal domain of pol b. DNA pol b specializes in single nucleotide gap filling and has reduced activity on larger gaps. Repair synthesis of larger gaps is likely preformed by another (other) polymerase(s), i.e. DNA pol d or e; although a role in long patch repair has been suggested. Increased DNA pol b activity is associated acquired resistance to platin-based therapy in brain tumors. Mechanistically, DNA pol b has been shown to confer resistance to chloroethylating, methylating, DNA cross linking agents and irradiation. DNA pol b is error prone compared to the other mammalian DNA polymerases. The higher error rates of pol b are in part due to a lack of a 3’ Æ 5’ proofreading exonuclease activity. Increased dependence on pol b, due to increased single bp gaps, may result in an accumulation of point mutations. Increases in DNA pol b activity have been associated with carcinogenesis and genomic instability.