The major goals of this project are to determine the molecular mechanisms of heteroduplex DNA base mismatch correction in Streptococcus pneumoniae and to assess the presence of related systems of generalized mismatch repair in other organisms. In S. pneumonia such repair occurs after DNA-mediated chromosomal transformation and after potentially mutagenic base substitution in DNA replication. Chromosomal mutations called hex block action of the repair system. Two unlinked genes, hexA and hexB, encode proteins implicated in the repair process. The genes have been cloned and their sequences determined. In addition to recognizing a mismatch, which can be an abnormal base pair or a short insertion/deletion, the system targets the strand to be corrected on the basis, apparently, of single-strand breaks bracketing the mismatch. These breaks can be a distance of kilobase or more from the mismatch.
One aim i s to ascertain whether the breaks in nascent DNA giving Okazaki fragments are responsible for targeting, and how these breaks, which appear to be present in the leading as well as the lagging strand being synthesized, are made. The possible role of the DNA glycosylases and AP-endonucleases in causing breaks in the leading strand will be investigated. During repair, a target strand segment containing the mismatch is apparently destroyed. The role of a pneumococcal DNA polymerase, which has been cloned, in restoring the DNA strand within the resulting gap will be tested. The HexA protein will be purified, with the help of an antibody directed to it, and the protein will be characterized with respect to its binding to ATP and to DNA mismatches. HexB will also be purified. A possible requirement for ATP hydrolysis by a complex of HexA and HexB scanning the DNA target for strand breaks and mismatches will be investigated. The anti-HexA antibody will be used to detect cross-reacting proteins in human cell extracts. Preliminary results indicate such a cross-reacting protein. This protein will be isolated and compared to HexA and to a possibly homologous protein encoded by a gene adjacent to dhfr. A human protein homologous to HexB will be similarly sought, and cDNAs encoding both homologs will be obtained. They will be used to assess the role of mismatch repair in preventing the accumulation of mutations that cause ageing and to test whether a genetic defect in the system results in the progeric disease of Werner's Syndrome.
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