DNA Repair; DNA Replication; Genetics; Mutagenesis; Radiation Oncology; Carcinogenesis
Therapeutic Radiology: Radiobiology | Therapeutic Radiology - Research
The focus of my research is genomic instability and how it leads to the mutations that result in human diseases such as cancer. The goal of one of the projects in my laboratory is to understand how DNA polymerases synthesize DNA accurately, and how they make mistakes that result in mutations. We study DNA mammalian DNA polymerase beta (Pol ß) because it was a relatively small, single subunit enzyme that could be easily manipulated and purified. My laboratory developed a method for assessing the activity and accuracy of rat Pol ß in bacteria. This led to the identification of a number of variants of Pol ß that synthesized DNA inaccurately. Our focus upon detailed biochemical characterization of variants of the Pol ß protein has shown that single amino acid residues that are quite distant from the active site of the enzyme are very important for accurate DNA synthesis. We found that many of these residues act at a distance in DNA positioning, nucleotide binding, and by influencing the rates of conformational changes that are important for accurate DNA synthesis. A second project is focused upon tumor-associated variants of Pol ß. Pol ß functions in base excision repair. The base excision repair system is responsible for removal of at least 10,000 DNA lesions per day. Interestingly, the Pol ß gene appears to be mutated in a large percentage of tumors. We have found that expression of the tumor-associated variants in mouse cells results in cellular transformation that has a mutational mechanism. We have shown that some of the Pol ß variants synthesize DNA inaccurately and others have no catalytic activity, but can bind to DNA and interfere with its repair. Our results suggest that the variants compromise base excision repair and lead to the induction of mutations, and suggest that base excision repair itself is a tumor suppressor mechanism. These studies impact our fundamental understanding of DNA repair and have the potential to lead to the design of targeted cancer therapeutics.
Specialized Terms: Genome Stability; Mutagenesis; DNA Replication; DNA Repair; Carcinogenesis
Extensive Research DescriptionA major goal of the research conducted in my laboratory is to study the relationship between mutations and cancer. Therefore we concentrate our efforts on studying the mechanism of mutagenesis by a DNA polymerase, polymerase § (Pol §). We employ genetic screens to identify amino acid residues of Pol § that function in promoting the fidelity of DNA synthesis. The Pol § mutant proteins identified in these screens are then analyzed using kinetics to determine the mechanism(s) Pol § employs to synthesize DNA accurately.
We also study the cellular role of Pol § and have shown that Pol § participates in base excision repair and in the process of meiosis. We are pursuing these studies to determine how Pol § makes errors during base excision repair and to identify the role of Pol § in meiosis.Pol § IgG stains discrete foci on mouse chromosome homologs during prophase I of meiosis in mouse spermatocytes. Mouse spermatocytes in early prophase. A, B) Nucleus in late zygonema. Pol §-stained nuclei (red) are on the left; Pol § and Cor I-stained nuclei (white) on the right. C) Nucleus in mid-pachynema. Merged image of Pol § and Cor I-stained nucleus. D) Nucleus in late pachynema and proceeding into diplonema. Merged image of Pol § and Cor I-stained nucleus. Control experiments done in parallel with equivalent chromosomal spreads using preimmune serum or Pol §-depleted IgG fractions yielded no detectable staining (data not shown).
- Kidane D, Murphy DL, Sweasy JB. Accumulation of abasic sites induces genomic instability in normal human gastric epithelial cells during Helicobacter pylori infection. Oncogenesis. 2014; 3:e128
- Sjolund A, Nemec AA, Paquet N, Prakash A, Sung P, Doublié S, Sweasy JB. A germline polymorphism of thymine DNA glycosylase induces genomic instability and cellular transformation. PLoS Genet. 2014; 10(11):e1004753
- Towle-Weicksel JB, Dalal S, Sohl CD, Doublié S, Anderson KS, Sweasy JB. Fluorescence resonance energy transfer studies of DNA polymerase ß: the critical role of fingers domain movements and a novel non-covalent step during nucleotide selection. J Biol Chem. 2014; 289(23):16541-50
- Senejani AG, Liu Y, Kidane D, Maher SE, Zeiss CJ, Park HJ, Kashgarian M, McNiff JM, Zelterman D, Bothwell AL, Sweasy JB. Mutation of POLB causes lupus in mice. Cell Rep. 2014; 6(1):1-8
- Eckenroth BE, Towle-Weicksel JB, Sweasy JB, Doublié S. The E295K cancer variant of human polymerase ß favors the mismatch conformational pathway during nucleotide selection. J Biol Chem. 2013; 288(48):34850-60
- Galick HA, Kathe S, Liu M, Robey-Bond S, Kidane D, Wallace SS, Sweasy JB. Germ-line variant of human NTH1 DNA glycosylase induces genomic instability and cellular transformation. Proc Natl Acad Sci U S A. 2013 ;110(35):14314-9.
- Yamtich J, Nemec AA, Keh A, Sweasy JB. A germline polymorphism of DNA polymerase beta induces genomic instability and cellular transformation. PLoS Genet. 2012;8(11):e1003052. doi: 10.1371/journal.pgen.1003052.