Characterization of the rad14-2 mutant of Saccharomyces cerevisiae: implications for the recognition of UV photoproducts by the Rad14 protein.
The RAD14 gene of Saccharomyces cerevisiae is required for the incision step of the nucleotide excision repair process. The Rad14 protein can bind zinc, possesses a potential zinc finger DNA binding domain and has been shown to bind specifically to damaged DNA. Differences in UV sensitivity exist between a rad14 deletion strain and a putative rad14 point mutant, the point mutant being more resistant to UV than the deletion strain. Here, we confirm that the rad14 deletion strain repairs neither UV-induced cyclobutane pyrimidine dimers (CPDs) nor endonuclease III-sensitive damage sites, whereas the point mutant cannot repair the former but can repair the latter. From this it can be inferred that the point mutant produces an altered protein product allowing recognition of endonuclease III sensitive sites but not CPDs. To investigate this, the rad14 mutant allele was sequenced. It contained two GC-AT transition mutations when compared to the wild-type RAD14 gene sequence. When the rad14 point mutant sequence is translated, alterations within the putative zinc finger binding domain are observed, with one of the cysteine residues of the zinc binding motif being replaced by tyrosine. This suggests that alterations within the zinc finger binding domain of the Rad14 protein cause changes to the damage recognition properties of the protein. The use of the Rad14 protein from the point mutant should assist in experiments investigating the in vitro binding properties of the Rad14 protein to different types of DNA damage.
Jones
G W
GW
School of Biological Sciences, University College of Swansea, Singleton Park, U.K.
Reed
S H
SH
Waters
R
R
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004260:DNA Repair / Q000235:genetics / Q000502:physiology*; D045643:DNA Repair Enzymes; D005656:Fungal Proteins / Q000502:physiology*; D008969:Molecular Sequence Data; D010641:Phenotype; D011740:Pyrimidine Dimers / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D017385:Sequence Homology; D014466:Ultraviolet Rays
D005656:Fungal Proteins; D011740:Pyrimidine Dimers; C072975:RAD14 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D045643:DNA Repair Enzymes