Multiple pathways for Okazaki fragment processing in eukaryotes = 진핵생물의 오카자키 단편 처리 과정의 다양성

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Okazaki fragment processing is hot spot for initiation of genome instability in eukaryotes. The principal mechanism of Okazaki fragment processing is via generation of flaps, which have a potential to form a variety of structures in their sequence context. These structures are properly processed by multiple layers of redundant pathways, which network with each other in intricate ways. Here, we elucidate the role of Vts1 and Rad52 in Okazaki fragment processing. The non-essential $\sl{VTS1}$ gene of $\sl{Saccharomyces cerevisiae}$ is highly conserved in eukaryotes and encodes a sequence- and structure-specific RNA binding protein. The Vts1 protein has been implicated in posttranscriptional regulation of a specific set of mRNAs that contains its binding site at their 3’-untranslated region. In this study, we identified $\sl{VTS1}$ as a multi-copy suppressor of $\sl{dna2-K1080E}$, a lethal mutant allele of $\sl{DNA2}$ that lacks DNA helicase activity. The suppression was allele-specific, since overexpression of Vts1 did not suppress the temperature-dependent growth defects of $\sl{dna2Δ405N}$, devoid of the N-terminal 405 amino acid residues. Purified recombinant Vts1 stimulated the endonuclease activity of wild-type Dna2, but not the endonuclease activity of Dna2Δ405N, indicating that the activation requires the N-terminal domain of Dna2. Stimulation of Dna2 endonuclease activity by Vts1 appeared to be the direct cause of suppression, since the multi-copy expression of Dna2-K1080E suppressed the lethality observed with its single-copy expression. We found that $\sl{vts1Δ dna2Δ405N}$ and $\sl{vts1Δ dna2-7 }$double mutant cells displayed synergistic growth defects, in support of a functional interaction between two genes. Our results provide both $\sl{in vivo}$ and $\sl{in vitro}$ evidence that Vts1 is involved in lagging strand synthesis by modulating the Dna2 endonuclease activity that plays an essential role in Okazaki fragment processing. Rad52 is a key prot...
Seo, Yeon-Sooresearcher서연수researcher
한국과학기술원 : 생명과학과,
Issue Date
455361/325007  / 020057487

학위논문(박사) - 한국과학기술원 : 생명과학과, 2010.08, [ ix, 131 p. ]


Vts1; Dna2; DNA recombination; DNA replication; Rad52; Rad52; Vts1; Dna2; DNA 상동재조합; DNA 복제

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