The molecular mechanisms of mutagenesis will be examined at the enzymological level, utilizing recently developed techniques for manipulation and analysis of DNA. In one approach, recombinant DNA molecules, bearing genetic markers of Haemophilus influenzae, will be treated with mutagenic agents and then incubated with cell-free extracts under conditions in in vitro replication and/or repair. Establishment of mutations will be monitored by a sensitive genetic assay that utilizes transformation of H influenzae. This in vitro mutagenesis system will be used to purify proteins involved in mutagenesis, with emphasis on inducible activities required for certain types of mutagenesis in vivo. In another approach defined template primers, prepared by annealing [5'-32P] restriction fragments to single-stranded templates, will be incubated with purified DNa polymerases and other proteins that are potentially involved in mutagenesis. The lengths of elongated primers will be analyzed by high resolution gel electrophoresis/autoradiography. The accuracy of DNA synthesis will be assessed by measurement of chian elongation in the presence of only 3 of the 4 dNTPs. DNA synthesis of mutagen-damaged templates will be characterized to determine which nucleotides, if any, are inserted opporiste specific lesions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM030590-04
Application #
3278373
Study Section
(MG)
Project Start
1982-05-01
Project End
1987-04-30
Budget Start
1985-05-01
Budget End
1986-04-30
Support Year
4
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Baylor College of Medicine
Department
Type
Schools of Medicine
DUNS #
074615394
City
Houston
State
TX
Country
United States
Zip Code
77030
Maldonado-Rodriguez, R; Driggers, P H; Beattie, K L (1991) Genetic assay of misincorporation. Mutat Res 251:201-16
Maldonado-Rodriguez, R; Espinosa-Lara, M; Beattie, K L (1991) Influence of neighboring base sequence on mutagenesis induced by in vitro misincorporation in the lacI gene of Escherichia coli. Mutat Res 251:217-26
Maldonado-Rodriguez, R; Beattie, K L (1991) In vitro mutagenesis in the lacI gene of Escherichia coli: fate of 3'-terminal mispairs versus internal base mispairs in a transfection assay. Mutat Res 247:5-18
Lai, M D; Beattie, K L (1988) Influence of DNA sequence on the nature of mispairing during DNA synthesis. Biochemistry 27:1722-8
Beattie, K L; Logsdon, N J; Anderson, R S et al. (1988) Gene synthesis technology: recent developments and future prospects. Biotechnol Appl Biochem 10:510-21
Driggers, P H; Beattie, K L (1988) Effect of pH on the base-mispairing properties of 5-bromouracil during DNA synthesis. Biochemistry 27:1729-35
Lai, M D; Beattie, K L (1988) Influence of divalent metal activator on the specificity of misincorporation during DNA synthesis catalyzed by DNA polymerase I of Escherichia coli. Mutat Res 198:27-36
Anderson, R S; Lawrence, C B; Wilson, S H et al. (1987) Genetic relatedness of human DNA polymerase beta and terminal deoxynucleotidyltransferase. Gene 60:163-73
Revich, G G; Beattie, K L (1986) Utilization of 1,N6-etheno-2'-deoxyadenosine 5'-triphosphate during DNA synthesis on natural templates, catalyzed by DNA polymerase I of Escherichia coli. Carcinogenesis 7:1569-76
Hillebrand, G G; Beattie, K L (1985) Influence of template primary and secondary structure on the rate and fidelity of DNA synthesis. J Biol Chem 260:3116-25