Mismatch repair has recently attracted a great deal of attention due to the discovery that mutations in mismatch repair enzymes are linked to hereditary colon cancer. E. Coli has an elaborate line of defense against the deleterious effects of mismatches. Decades of work on the E. Coli systems has significantly advanced the understanding of these important enzymes. However, even with years of research on the bacterial enzymes, many important and unanswered questions concerning DNA repair enzymes remain. Indeed, the factors influencing the recognition of DNA damage including the effects of sequence environment surrounding the damage are still unclear. The E. Coli enzyme, MutY is important in the prevention of mutations caused by mismatches that arise due to miscoding from oxidative damage to DNA. MutY acts as an adenine glycosylase to remove an undamaged adenine paired with 7,8-dihydro-8-oxoguanine (OG). Very little mechanistic work has focused on glycosylases that act on purine bases. An unusual aspect of MutY is that it is a [4Fe-4S] cluster-containing protein and has significant homology to the DNA repair enzyme, endonuclease III. The possibility that the [4Fe-4S] cluster domain could represent a new motif for mismatch recognition and repair is indeed quite intriguing. Thus, MutY is not only functionally important in maintaining high fidelity DNA replication, it also has unique structural and mechanistic properties. The goal of the proposed research is to provide a detailed understanding of the structural and functional properties of MutY. The initial focus will be determining the properties of G:A and OG:A mismatch containing substrates which are critical for recognition and repair by MutY. In particular, the influence of the sequence environment around the G:A mismatch will be determined as well as the a determination of the origin of sequence dependent effects. Modified G:A and OG:A containing substrates will be prepared to determine the important functional groups on the G/OG or the A that are required for efficient mismatch recognition. Novel inhibitors for MutY will be prepared to provide mechanistic information and as tools for biochemical and spectroscopic studies of the enzyme- inhibitors complex. A solid understanding of the properties of the DNA substrates for MutY will provide the subsequent basis for investigation of the properties of MutY-DNA complexes with a particular emphasis on elucidation of the role of the unusual metal center in the enzyme. These experiments will focus on determining whether or not the [4Fe-4S] cluster is intimately involved in mismatch recognition and DNA binding.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29CA067985-06
Application #
2895322
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Okano, Paul
Project Start
1995-07-01
Project End
2000-06-30
Budget Start
1999-07-01
Budget End
2000-06-30
Support Year
6
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Utah
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Banda, Douglas M; Nuñez, Nicole N; Burnside, Michael A et al. (2017) Repair of 8-oxoG:A mismatches by the MUTYH glycosylase: Mechanism, metals and medicine. Free Radic Biol Med 107:202-215
Manlove, Amelia H; McKibbin, Paige L; Doyle, Emily L et al. (2017) Structure-Activity Relationships Reveal Key Features of 8-Oxoguanine: A Mismatch Detection by the MutY Glycosylase. ACS Chem Biol 12:2335-2344
Wickramaratne, Susith; Banda, Douglas M; Ji, Shaofei et al. (2016) Base Excision Repair of N(6)-Deoxyadenosine Adducts of 1,3-Butadiene. Biochemistry 55:6070-6081
Woods, Ryan D; O'Shea, Valerie L; Chu, Aurea et al. (2016) Structure and stereochemistry of the base excision repair glycosylase MutY reveal a mechanism similar to retaining glycosidases. Nucleic Acids Res 44:801-10
Brinkmeyer, Megan K; David, Sheila S (2015) Distinct functional consequences of MUTYH variants associated with colorectal cancer: Damaged DNA affinity, glycosylase activity and interaction with PCNA and Hus1. DNA Repair (Amst) 34:39-51
Ono, Toshikazu; Wang, Shenliang; Koo, Chi-Kin et al. (2012) Direct fluorescence monitoring of DNA base excision repair. Angew Chem Int Ed Engl 51:1689-92
Brinkmeyer, Megan K; Pope, Mary Ann; David, Sheila S (2012) Catalytic contributions of key residues in the adenine glycosylase MutY revealed by pH-dependent kinetics and cellular repair assays. Chem Biol 19:276-86
Engstrom, Lisa M; Partington, Olga A; David, Sheila S (2012) An iron-sulfur cluster loop motif in the Archaeoglobus fulgidus uracil-DNA glycosylase mediates efficient uracil recognition and removal. Biochemistry 51:5187-97
Raetz, Alan G; Xie, Yali; Kundu, Sucharita et al. (2012) Cancer-associated variants and a common polymorphism of MUTYH exhibit reduced repair of oxidative DNA damage using a GFP-based assay in mammalian cells. Carcinogenesis 33:2301-9
Michelson, Anna Zhachkina; Rozenberg, Aleksandr; Tian, Yuan et al. (2012) Gas-phase studies of substrates for the DNA mismatch repair enzyme MutY. J Am Chem Soc 134:19839-50

Showing the most recent 10 out of 13 publications