Involvement of Vts1, a structure-specific RNA-binding protein, in Okazaki fragment processing in yeast.
The non-essential VTS1 gene of Saccharomyces cerevisiae is highly conserved in eukaryotes and encodes a sequence- and structure-specific RNA-binding protein. The Vts1 protein has been implicated in post-transcriptional regulation of a specific set of mRNAs that contains its-binding site at their 3'-untranslated region. In this study, we identified VTS1 as a multi-copy suppressor of dna2-K1080E, a lethal mutant allele of DNA2 that lacks DNA helicase activity. The suppression was allele-specific, since overexpression of Vts1 did not suppress the temperature-dependent growth defects of dna2Delta405N 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 Dna2Delta405N, 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 vts1Delta dna2Delta405N and vts1Deltadna2-7 double mutant cells displayed synergistic growth defects, in support of a functional interaction between two genes. Our results provide both in vivo and 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.
Lee
Chul-Hwan
CH
Center for DNA Replication and Genome Instability, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.
Shin
Yong-Keol
YK
Phung
Thi Thu Huong
TT
Bae
Jae Seok
JS
Kang
Young-Hoon
YH
Nguyen
Tuan Anh
TA
Kim
Jeong-Hoon
JH
Kim
Do-Hyung
DH
Kang
Min-Jung
MJ
Bae
Sung-Ho
SH
Seo
Yeon-Soo
YS
D000483:Alleles; D002467:Cell Nucleus / Q000737:chemistry; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D045585:Flap Endonucleases / Q000378:metabolism; D009154:Mutation; D016601:RNA-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D013489:Suppression, Genetic
D004268:DNA-Binding Proteins; C022262:Okazaki fragments; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; C508663:Vts1 protein, S cerevisiae; D004247:DNA; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases; C468323:RAD27 protein, S cerevisiae; D004265:DNA Helicases; C410222:DNA2 protein, S cerevisiae