Abstract

This project will investigate DNA base mismatch correction in Streptococcus pneumoniae. Such repair occurs after DNA-mediated genetic transformation and after potentially mutagenic base substitution in DNA replication. In transformation, single-site markers are integrated with different efficiencies because a system, called Hex, recognizes mismatches in the initial heteroduplex product of integration and removes the donor portion with a frequency depending on the mismatched base pair formed. Hex mutants give only high integration efficiency. The molecular mechanism of the Hex system will be determined in vivo by following the biochemical fate of a homogeneous DNA fragment containing the amylomaltase gene. this gene has been extensively analyzed and cloned. A newly developed system for self-cloning in S. pneumoniae will allow amplification of the repair response and analysis of mismatch repair in plasmid transformation. The investigation of plasmid transformation should clarify several novel genetic recombinatory processes in addition to mismatch repair. By design of a suitable substrate and use of cell extracts lacking nonspecific nucleases, we hope to demonstrate mismatch repair in vitro. The enzymatic mechanism of the repair process could then be analyzed. Of particular importance is how the system recognizes and excludes the donor contribution to the mismatch. Strains that lack the mismatch repair system show higher spontaneous mutation rates. The system apparently repairs DNA replication errors by recognizing base mismatches and removing the wrong base in the mascent strand. Mismatch repair, as indicated by the occurence of gene conversion, is widespread among living cells. The proficiency of this system could be a major factor in determining spontaneous mutation rates. If ageing is due to the accumulation of mutations, mismatch repair could be important for longevity and for the prevention of age-related diseases such as cancer and heart disease. An in vitro assay will make the study of mismatch repair accessible also in human cells.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI014885-08
Application #
3125905
Study Section
(MG)
Project Start
1978-04-01
Project End
1987-03-31
Budget Start
1985-04-01
Budget End
1986-03-31
Support Year
8
Fiscal Year
1985
Total Cost
Indirect Cost

  Institution

Name
Associated University-Brookhaven National Lab
Department
Type
DUNS #
City
Upton
State
NY
Country
United States
Zip Code
11973

  Publications

Lacks, S A; Ayalew, S; de la Campa, A G et al. (2000) Regulation of competence for genetic transformation in Streptococcus pneumoniae: expression of dpnA, a late competence gene encoding a DNA methyltransferase of the DpnII restriction system. Mol Microbiol 35:1089-98
Zhang, Y B; Ayalew, S; Lacks, S A (1997) The rnhB gene encoding RNase HII of Streptococcus pneumoniae and evidence of conserved motifs in eucaryotic genes. J Bacteriol 179:3828-36
Lacks, S A (1997) Cloning and expression of pneumococcal genes in Streptococcus pneumoniae. Microb Drug Resist 3:327-37
Lacks, S A; Greenberg, B; Lopez, P (1995) A cluster of four genes encoding enzymes for five steps in the folate biosynthetic pathway of Streptococcus pneumoniae. J Bacteriol 177:66-74
Kumaresan, K R; Springhorn, S S; Lacks, S A (1995) Lethal and mutagenic actions of N-methyl-N'-nitro-N-nitrosoguanidine potentiated by oxidized glutathione, a seemingly harmless substance in the cellular environment. J Bacteriol 177:3641-6
Diaz, A; Lacks, S A; Lopez, P (1994) Multiple roles for DNA polymerase I in establishment and replication of the promiscuous plasmid pLS1. Mol Microbiol 14:773-83
Lopez, P; Lacks, S A (1993) A bifunctional protein in the folate biosynthetic pathway of Streptococcus pneumoniae with dihydroneopterin aldolase and hydroxymethyldihydropterin pyrophosphokinase activities. J Bacteriol 175:2214-20
Diaz, A; Pons, M E; Lacks, S A et al. (1992) Streptococcus pneumoniae DNA polymerase I lacks 3'-to-5' exonuclease activity: localization of the 5'-to-3' exonucleolytic domain. J Bacteriol 174:2014-24
Diaz, A; Lacks, S A; Lopez, P (1992) The 5' to 3' exonuclease activity of DNA polymerase I is essential for Streptococcus pneumoniae. Mol Microbiol 6:3009-19
Chen, J D; Lacks, S A (1991) Role of uracil-DNA glycosylase in mutation avoidance by Streptococcus pneumoniae. J Bacteriol 173:283-90

Showing the most recent 10 out of 27 publications