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J. Bacteriol., 08 1995, 4402-4409, Vol 177, No. 15
Copyright © 1995, American Society for Microbiology

Molecular dissection of mutations in the Bacillus subtilis spore photoproduct lyase gene which affect repair of spore DNA damage caused by UV radiation

P Fajardo-Cavazos and WL Nicholson
Department of Microbiology and Immunology, University of North Texas Health Science Center, Fort Worth 76107, USA.

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 UV-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.(ABSTRACT TRUNCATED AT 250 WORDS)


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