The dark repair of damaged DNA can involve a based excision pathway in which glycosylases specific for damaged bases hydrolyze the N-glycosidic bond between the base and the sugar and endonucleases remove the resulting deoxyribose-5-phosphate moiety. Particular attention will be given to the mechanisms of the reactions catalyzed by glycosylases which remove damaged pyrimidine bases, with particular emphasis being placed on the reaction catalyzed by uracil-DNA glycosylase. Model studies will be performed on various deoxynucleosides to determine whether saturation of the 5,6-double bond of the pyrimidine (by nucleophilic addition in the case of uracil) allows an acid-catalyzed mechanisms in which an imonium ion is formed between the base and sugar and the sugar ring is opened; these studies will rely on kinetic isotope effects, elemental substitution in the sugar ring, and 17O NMR spectroscopy. Once the model systems are understood, the mechanisms of the reaction catalyzed by uracil-DNA glycosylase will be examined. The mechanisms of strand cleavage at aldehydic abasic sites catalyzed by both class I and class II AP endonucleases will also be studied. Exonuclease III and endonuclease IV hydrolytically cleave the 5'- phosphodiester bond involving an aldehydic abasic site, and stereochemical studies will be performed to establish mechanistic relationships between these enzymes. Endonucleases III and UV endonuclease V catalyze both a damaged base-DNA glycosylase reaction and cleavage of the 3'-phosphodiester bond involving an aldehydic abasic site by a beta-elimination reaction. Studies of the chemical reactivity of aldehydic abasic sites will be performed so that the beta-elimination reactions catalyzed by these enzymes can be better understood. The relationships between the enzymatic activities and mechanistic details of both activities will be studied by measuring kinetic isotope effects and studying the processing of substrate analogs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034572-10
Application #
3285831
Study Section
Biochemistry Study Section (BIO)
Project Start
1984-08-01
Project End
1994-08-31
Budget Start
1993-08-01
Budget End
1994-08-31
Support Year
10
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Maryland College Park
Department
Type
Schools of Earth Sciences/Natur
DUNS #
City
College Park
State
MD
Country
United States
Zip Code
20742
Landro, J A; Gerlt, J A; Kozarich, J W et al. (1994) The role of lysine 166 in the mechanism of mandelate racemase from Pseudomonas putida: mechanistic and crystallographic evidence for stereospecific alkylation by (R)-alpha-phenylglycidate. Biochemistry 33:635-43
Mitra, B; Gerlt, J A; Babbitt, P C et al. (1993) A novel structural basis for membrane association of a protein: construction of a chimeric soluble mutant of (S)-mandelate dehydrogenase from Pseudomonas putida. Biochemistry 32:12959-67
Petsko, G A; Kenyon, G L; Gerlt, J A et al. (1993) On the origin of enzymatic species. Trends Biochem Sci 18:372-6
Gerlt, J A; Gassman, P G (1993) Understanding the rates of certain enzyme-catalyzed reactions: proton abstraction from carbon acids, acyl-transfer reactions, and displacement reactions of phosphodiesters. Biochemistry 32:11943-52
Mazumder, A; Gerlt, J A; Absalon, M J et al. (1991) Stereochemical studies of the beta-elimination reactions at aldehydic abasic sites in DNA: endonuclease III from Escherichia coli, sodium hydroxide, and Lys-Trp-Lys. Biochemistry 30:1119-26
Powers, V M; Koo, C W; Kenyon, G L et al. (1991) Mechanism of the reaction catalyzed by mandelate racemase. 1. Chemical and kinetic evidence for a two-base mechanism. Biochemistry 30:9255-63
Landro, J A; Kallarakal, A T; Ransom, S C et al. (1991) Mechanism of the reaction catalyzed by mandelate racemase. 3. Asymmetry in reactions catalyzed by the H297N mutant. Biochemistry 30:9274-81
Tsou, A Y; Ransom, S C; Gerlt, J A et al. (1990) Mandelate pathway of Pseudomonas putida: sequence relationships involving mandelate racemase, (S)-mandelate dehydrogenase, and benzoylformate decarboxylase and expression of benzoylformate decarboxylase in Escherichia coli. Biochemistry 29:9856-62
Tsou, A Y; Ransom, S C; Gerlt, J A et al. (1989) Selection and characterization of a mutant of the cloned gene for mandelate racemase that confers resistance to an affinity label by greatly enhanced production of enzyme. Biochemistry 28:969-75
Ransom, S C; Gerlt, J A; Powers, V M et al. (1988) Cloning, DNA sequence analysis, and expression in Escherichia coli of the gene for mandelate racemase from Pseudomonas putida. Biochemistry 27:540-5

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