DNA helicase III of Saccharomyces cerevisiae, encoded by YER176w (HEL1), highly unwinds covalently closed, circular DNA in the presence of a DNA topoisomerase and yRF-A.
Previously, we have purified and characterized DNA helicase III from the yeast Saccharomyces cerevisiae [Shimizu, K. and Sugino, A. (1993) J. Biol. Chem. 268, 9578-9584]. Here, we have further characterized DNA helicase III activity. It was found that the combined action of the helicase III, yeast DNA topoisomerase I (yTop I), and yeast RPA protein on a covalently closed, circular DNA generates a highly underwound DNA species that has been called form I* or form U. Furthermore, these underwound structures can be accessed by yeast DNA polymerase I (alpha)-primase to initiate DNA synthesis. These reactions mimic in vivo initiation of chromosomal DNA replication. In order to clone the gene encoding DNA helicase III, a partial amino acid sequence of the purified DNA helicase III polypeptide was determined. Using a mix oligonucleotides synthesized based on the amino acid sequence of the helicase, we cloned the gene encoding the helicase III and found it to be identical to YER176W (HEL1) on chromosome V. The amino acid sequence predicted from the nucleotide sequence of the gene has conserved DNA helicase domains that are highly homologous to those of DNA helicases required for DNA replication. However, complete deletion of the gene from the chromosome did not result in any growth defect, suggesting that the gene product is not required for DNA synthesis or that it is functionally substituted by other helicase(s). Furthermore, the deletion strain does not exhibit sensitivity to any DNA-damaging reagents, although it is hypersensitive to calcofluor white, hygromycin, and papulacandin.
Kamimura
Y I
YI
Department of Biochemistry and Molecular Biology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871, Japan.
Kawasaki
Y
Y
Ohara
T
T
Sugino
A
A
D000595:Amino Acid Sequence; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004270:DNA, Circular / Q000737:chemistry* / Q000378:metabolism; D004271:DNA, Fungal / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid