The major goal of the project is to determine the molecular mechanism of heteroduplex DNA base mismatch correction in Streptococcus pneumoniae. Such repair occurs after DNA- mediated chromosomal transformation and after potentially mutagenic base substitution in DNA replication. A number of chromosomal mutations, called hex, block action of the repair system. Restriction enzyme analyses of DNA containing various hex mutations showed that two distinct genes, hexA and hexB were implicated in the repair process. The hexA gene was cloned in an S. pneumoniae host/vector system, and its product was shown to be a 90 kDa polypeptide. The nucleotide sequence of the hexA gene and surrounding DNA will be determined. This sequence will be examined for the presence of transcription and translation signals and open reading frames. Site-directed mutagenesis should identify the hexA gene and clarify the function of other elements or genes in the operon. The native forms of these gene products will be radioactively labeled and isolated, first from minicells of B. subtilis, then from cells of S. pneumoniae. Purification will be carried out be column fractionation with various chromatographic media. The polypeptide structure of the native proteins will be determined. A model heteroduplex substrate for mismatch repair will be constructed from ma1M gene DNA fragments cloned both in streptococcal plasmids and in E. coli single-strand phage vectors. This substrate will hopefully allow detection of mismatch repair functions in vitro with crude extracts and purified hex gene products. The gene encoding a DNA polymerase-exonuclease of S. pneumoniae was recently cloned, and site-directed mutants will be obtained to see if this enzyme participates in mismatch repair. These studies should lead to an understanding of the biochemical mechanism of the repair process. Further examination of two other modes of DNA interaction, plasmid genome transformation and chromosomal facilitation of plasmid transfer will be undertaken. The molecular mechanisms of these processes will be investigated with suitably constructed donor DNA molecules, and the effect of various rec and hex mutations on these novel modes of recombination will be ascertained. In addition, an investigation of recombinant plasmid instability and the mechanism of generation of deletions will continue.
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