Functional characterization of the fission yeast $pfh1^+$ gene, which encodes an essential 5' to 3' DNA helicase. = DNA 헬리케이즈 활성을 갖는 분열효모의 $pfh1^+$ 유전자의 기능 규명

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Schizosaccharomyces pombe $pfh1^{+}$ was isolated as a genetic suppressor of a temperature-sensitive (ts) mutant allele of $cdc24^{+}$ that functions with $dna2^{+}$ in Okazaki fragment processing. We determined enzymatic activities of the Pfh1 enzyme and genetic interactions between $pfh1^{+}$ and $dna2^{+}$. The ts phenotype of dna2-C2 mutant was suppressed when dna2-C2 was combined with pfh1-R20. The double mutant cells still retained the cold-sensitive (cs) phenotype of pfh1-R20. Overexpression of wild type $pfh1^{+}$ in this double mutant cell rescued the cs phenotype of pfh1-R20, but not the ts growth defect of dna2-C2. This result suggests either (1) that an allele-specific interaction between dna2-C2 and pfh1-R20 or (2) that inactivation of $pfh1^{+}$ is required for the rescue of defects associated with dna2-C2. The fact that Pfh1-R20 mutant protein purified displayed significantly reduced ATPase and helicase activities and the pfh1-R23 allele also rescued the ts growth defect of dna2-C2 argues for the latter possibility. The Pfh1 helicase activity was stimulated by fork structure and can use internal ssDNA as entry site. About 10-nt of ssDNA is needed for efficient loading of Pfh1 helicase and longer than 20-nt of ssDNA is no more needed. Pfh1 is not processive helicase and inhibited by RPA. The pol3 and cdc27 mutants (subunits of pol δ) were able to suppress the ts phenotype of dna2-C2 mutant. In contrast, combination of pfh1-R20 with pol3 or cdc27 resulted in synthetic lethal growth. The genetic interactions above suggest that Pfh1 plays a role in collaboration with Dna2 and pol δ during Okazaki fragment processing.
Advisors
Seo, Yeon-Sooresearcher서연수researcher
Description
한국과학기술원 : 생명과학과,
Publisher
한국과학기술원
Issue Date
2004
Identifier
237526/325007  / 000985117
Language
eng
Description

학위논문(박사) - 한국과학기술원 : 생명과학과, 2004.2, [ vi, 79 p. ]

Keywords

FISSION YEAST; POMBE; HELICASE; REPLICATION; 복제; pfh1; 효모; 헬리케이즈; OKAZAKI FRAGMENT

URI
http://hdl.handle.net/10203/27560
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=237526&flag=dissertation
Appears in Collection
BS-Theses_Ph.D.(박사논문)
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