Saccharomyces cerevisiae Hrq1 helicase activity is affected by the sequence but not the length of single-stranded DNA

Abstract

a b s t r a c tMutations in the human RecQ4 DNA helicase are associated with three different diseases characterized by genomic instability. To gain insight into how RecQ4 dysfunction leads to these pathologies, several groups have used the Saccharomyces cerevisiae RecQ4 homolog Hrq1 as an experimental model. Hrq1 displays many of the same functions as RecQ4 in vivo and in vitro. However, there is some disagreement in the literature about the effects of single-stranded DNA (ssDNA) length on Hrq1 helicase activity and the ability of Hrq1 to anneal complementary ssDNA oligonucleotides into duplex DNA. Here, we present a side-by-side comparison of Hrq1 and RecQ4 helicase activity, demonstrating that in both cases, long random-sequence 3 0 ssDNA tails inhibit DNA unwinding in vitro in a length-dependent manner. This appears to be due to the formation of secondary structures in the random-sequence ssDNA because Hrq1 preferentially unwound poly(dT)-tailed forks independent of ssDNA length. Further, RecQ4 is capable of ssDNA strand annealing and annealing-dependent strand exchange, but Hrq1 lacks these activities. These results establish the importance of DNA sequence in Hrq1 helicase activity, and the absence of Hrq1 strand annealing activity explains the previously identified discrepancies between S. cerevisiae Hrq1 and human RecQ4.

The Hrq1 helicase stimulates Pso2 translesion nuclease activity to promote DNA inter-strand crosslink repair

“…The data presented here and in our previous work (26) indicate that the Hrq1 helicase is involved in FA-independent ICL repair. This mirrors what is known about human RECQL4 in ICL repair in that recql4 mutant cells are sensitive to the ICL-inducing drug cisplatin (25), and RECQL4 does not belong to any of the known FA complementation groups (43,44).…”

“…Recent work on the RECQL4 homologs in Arabidopsis thaliana (23) and S. cerevisiae (3,24), both called Hrq1, demonstrates that they are involved in ICL repair, similar to RECQL4 (25). Furthermore, Hrq1 appears to be a bona fide RECQL4 homolog in vitro and in vivo, making it a good model for RECQL4-mediated DNA repair (3,24,26). Hrq1 is currently the only known protein to work with Pso2 at the post-incision step of ICL repair, but its mechanism of action is unclear.…”

Fibulin-3 Attenuates Phosphate-Induced Vascular Smooth Muscle Cell Calcification by Inhibition of Oxidative Stress

“…The present data shows that Fibulin-3 suppresses the increased expression of osteogenic transcription factors MSX2 and CBFA1 and of chondrogenic transcription factor SOX9 as well as ALPL expression and activity in VSMCs following high phosphate treatment. Previous studies similarly show that Fibulin-3 is able to reduce SOX9 expression and to inhibit chondrogenesis in bone marrow-derived mesenchymal stem cells as well as differentiation of chondrocytes [52, 53]. …”

TheSaccharomyces cerevisiaeHrq1 and Pif1 DNA helicases synergistically modulate telomerase activityin vitro

“…Samples were polished on a Resource S column and eluted in IEX buffer supplemented with NaCl using a gradient from 150 mM to 1 M. Samples were concentrated with Amicon Ultra-4 centrifugal filters with a 30K cutoff and stored at -80°C in storage buffer (25 mM HEPES (pH 7.5), 150 mM NaCl, 2 mM β -mercaptoethanol, 0.1 mM EDTA (pH 8.0), and 30% glycerol (w/v)). Hrq1 and Hrq1-K318A were overproduced in insect cells and purified as reported (15,61). All helicase preparations were tested for ATPase activity and the absence of contaminating nuclease activity before use in assays.…”