AtRECQ2, a RecQ-helicase homologue from Arabidopsis thaliana, is able to disrupt different recombinogenic DNA-structures in vitro

Abstract

RecQ helicases play an important role in the maintenance of genomic stability in pro- and eukaryotes. This is highlighted by the human genetic diseases Werner, Bloom's and Rothmund-Thomson syndrome, caused by respective mutations in three of the five human RECQ genes. The highest numbers of RECQ homologous genes are found in plants, e.g. seven in Arabidopsis thaliana. However, only limited information is available on the functions of plant RecQ helicases, and no biochemical characterization has been performed. Here, we demonstrate that AtRECQ2 is a (d)NTP-dependent 3'-->5' DNA helicase. We further characterized its basal properties and its action on various partial DNA duplexes. Importantly, we demonstrate that AtRECQ2 is able to disrupt recombinogenic structures: by disrupting various D-loop structures, AtRECQ2 may prevent non-productive recombination events on the one hand, and may channel repair processes into non-recombinogenic pathways on the other hand, thus facilitating genomic stability. We show that a synthetic partially mobile Holliday junction is processed towards splayed-arm products, possibly indicating a branch migration function for AtRECQ2. The biochemical properties defined in this work support the hypothesis that AtRECQ2 might be functionally orthologous to the helicase part of the human RecQ homologue HsWRN.

Biochemical Characterization of AtRECQ3 Reveals Significant Differences Relative to Other RecQ Helicases

“…S1) were prepared and incubated with both enzymes. As previously reported, AtRECQ2 was able to branch migrate the X12-HJ, a HJ composed of a homologous core of 12 bp flanked by arms of 19 bp (Kobbe et al, 2008). Comparative analysis of AtRECQ3 and AtRECQ3-K64M with this X12 junction did not reveal significant branch migration or unwinding (data not shown).…”

“…With previous biochemical characterizations of AtRECQ2 and the AtWRNexo protein (Plchova et al, 2003), we were able to substantiate the hypothesis of a functional homology between AtRECQ2 and the RecQ helicase part of HsWRN (Kobbe et al, 2008). In this work, we expanded the analysis of AtRECQ2 and compared its biochemical properties to those of the previously uncharacterized homolog AtRECQ3.…”

“…1A). The purification procedure for AtRECQ2 and AtRECQ2-K117M was described previously (Kobbe et al, 2008). The invariant Lys in the Walker A motif for AtRECQ3 was determined to be Lys-64 (counted without tags).…”

“…The invariant Lys in the Walker A motif for AtRECQ3 was determined to be Lys-64 (counted without tags). As an amino acid substitution at this position to Met was shown to inactivate the ATPase and therefore the helicase activity (Brosh et al, 1999;Kobbe et al, 2008), we used AtRECQ3-K64M preparations as a negative control. Like the AtRECQ2 proteins, AtRECQ3 and AtRECQ3-K64M (total calculated mass with affinity tags 86.0 kD) were overexpressed in E. coli and purified by consecutive nickel-immobilized metal ion affinity chromatography and calmodulin affinity chromatography (Fig.…”

“…Previously, we were able to characterize basic biochemical properties of the helicase RECQ2 of the model plant Arabidopsis (Kobbe et al, 2008). In this study, we chose to study AtRECQ3 as a RecQ helicase that differs in the domain structure from AtRECQ2, lacking the HRDC domain and the winged helix subdomain of the RQCt domain (Fig.…”