MOLECULAR DISSECTION OF MUTATIONS IN THE BACILLUS-SUBTILIS SPORE PHOTOPRODUCT LYASE GENE WHICH AFFECT REPAIR OF SPORE DNA-DAMAGE CAUSED BY UV-RADIATION

In response to UV irradiation, Bacillus subtilis spore DNA accumulates the unique thymine dimer 5-thyminyl-5,6-dihydrothymine, or spore photoproduct (SP), SP is broken down into monomers during spore germination by the product of the spl gene which has been proposed to encode the enzyme SP lyase. The wild-type spl gene was cloned by complementation of a mutation designated spl-1; the putative spl gene product is a 40-kDa protein whose deduced amino acid sequence contains regions homologous to DNA photolyases. During phenotypic characterization of spl subclones using transformation crosses between the cloned wild-type spl gene and an spl-1 mutant recipient, in addition to the expected transformant classes exhibiting UV-resistant (type I) and W-sensitive (type III) spores, an additional recombinant class was observed (called type II), spores of which exhibited slower germination kinetics following UV irradiation. The results suggested that the spl-1 allele consisted of at least two separable mutations, The DNA region which could rescue the spl-1 allele was localized to a 511-bp region within the spl coding sequence; this region was amplified from the spl-1 mutant chromosome by PCR and sequenced, The region contained two amino acid substitutions, an Arg replacing Gly-168 (G168R) and an Asp replacing Gly-242 (G242D) in the deduced SP lyase sequence, as well as 18 silent mutations, PCR amplification of chromosomal DNA from a selected type II recombinant and sequence analysis of the amplification product confirmed that recombination had indeed occurred between codons 168 and 242 and further localized the point of crossover by using the 18 silent mutations as molecular markers throughout the region, By in vitro mutagenesis, alleles of spl containing all combinations of single and double amino acid substitutions were introduced into the cloned wild-type spl gene. When integrated into the B. subtilis chromosome at the amyE locus, it was observed that although both amino acid substitutions contribute to the spl-1 phenotype, the G168R mutation exerted a much greater effect than did the G242D mutation,