Involvement of Vts1, a structure-specific RNA-binding protein, in Okazaki fragment processing in yeast

Abstract

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 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 vts1Δ dna2Δ405N and vts1Δdna2-7 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.

Rad52/Rad59-dependent Recombination as a Means to Rectify Faulty Okazaki Fragment Processing

“…This finding suggests that stimulation of the nuclease activity of Rad27 (Mgs1 and Mus81), Dna2 (RPA), or both (Vts1, Mph1, and Rad27) might not be the only mechanism by which suppression occurred (17, 58 -60, 64, 84). Previously, it was shown that the overexpression of nuclease-attenuated (34) or helicase-negative mutant Dna2 enzymes (60) or of wild type Rad27 (21) allows several dna2 mutant cells to grow, indicating that the enhanced nuclease activity of Dna2 or Fen1 FIGURE 7. Rad59 is essential for Rad52-mediated suppression of dna2-K1080E.…”

“…Alternatively, these levels of stimulation may not be enough to rescue dna2-K1080E. We, however, prefer the first possibility for the following reason: in many cases, the 5-10-fold stimulation of either Dna2 or Rad27 is sufficient to suppress growth defects of dna2 alleles (58,60,84). Therefore, we believe that the underlying mechanism for the Rad52-mediated suppression of dna2-K1080E is due to the increased levels of recombination events.…”

“…Direct Stimulation of Endonuclease Activity of Dna2 and Rad27 by Rad52 Is Not the Underlying Mechanism for the Suppression of dna2-K1080E-The simplest explanation for the suppression mechanism could be the direct stimulation of nuclease activity of either Dna2 or Rad27 or both by Rad52, as Rad27 and nuclease-proficient Dna2K1080E/Dna2⌬405N enzymes, upon overexpression, were able to suppress the lethal phenotype of dna2-K1080E (21,31,33,60). Alternatively, the Rad52-mediated suppression of dna2 mutations could be explained by efficient recombination-mediated repair of dam- dna2⌬405N cells (indicated as dna2⌬405N dna2⌬405NϩRAD51, and dna2⌬405NϩRAD52, respectively).…”

The Trans-autostimulatory Activity of Rad27 Suppresses dna2 Defects in Okazaki Fragment Processing

“…Alkaline Protein Extraction from Yeast Cells-Expression levels of proteins in yeast were determined using the alkaline protein extraction as described previously (37). The dan2⌬405N mutant cells harboring pRS325-ADH1-Rad27-FLAG (or other expression vectors for mutant derivatives of Rad27) were grown to saturation.…”

“…The samples were then subjected to electrophoresis in a 10% SDS-PAGE gel. Gels were Coomassie-stained and analyzed by Western blotting as described previously (37).…”