Multimerization domains are associated with apparent strand exchange activity in BLM and WRN DNA helicases

Abstract

BLM and WRN are members of the RecQ family of DNA helicases that act to suppress genome instability and cancer predisposition. In addition to a RecQ helicase domain, each of these proteins contains an N-terminal domain of approximately 500 amino acids (aa) that is incompletely characterized. Previously, we showed that the N-terminus of Sgs1, the yeast ortholog of BLM, contains a physiologically important 200 aa domain (Sgs1103–322) that displays single-stranded DNA (ssDNA) binding, strand annealing (SA), and apparent strand-exchange (SE) activities in vitro. Here we used a genetic assay to search for heterologous proteins that could functionally replace this domain of Sgs1 in vivo. In contrast to Rad59, the oligomeric Rad52 protein provided in vivo complementation, suggesting that multimerization is functionally important. An N-terminal domain of WRN was also identified that could replace Sgs1103–322 in yeast. This domain, WRN235–526, contains a known coiled coil and displays the same SA and SE activities as Sgs1103–322. The coiled coil domain of WRN235–526 was found to be required for both its in vivo activity and its in vitro SE activity. Based on this result, a potential coiled coil was identified within Sgs1103–322. This 25 amino acid region was similarly essential for wt Sgs1 activity in vivo and was replaceable by a heterologous coiled coil. Taken together, the results indicate that a coiled coil and a closely-linked apparent SE activity are conserved features of the BLM and WRN DNA helicases.

RPA phosphorylation regulates DNA resection

“…These RPA variants failed to stimulate helicase initiation (Figure 3E). More strikingly, all three variants-but not wt RPA-induced the intrinsic strand-switching activity previously reported for BLM and other RecQ helicases (Figure 3D,F and S3D) (Chen and Brill, 2014;Harami et al, 2017;Klaue et al, 2013;Wang et al, 2015;Yodh et al, 2009). Strand-switching refers to BLM alternating between translocation on either the Watson or Crick ssDNA strands (Wang et al, 2015;Yodh et al, 2009).…”

Mechanism of Bloom syndrome complex assembly required for double Holliday junction dissolution and genome stability

“…This is considerably larger than the predicted size of a complex containing one subunit of each protein (355 kDa). Because there is previous evidence that BLM forms dimers or multimers ( 24 – 27 ), we sought to determine whether BLM might induce an increase in the stoichiometry of other subunits in the BS complex. To do this, we carried out coexpression and purification of two TopoIIIα-RMI1-RMI2 complexes, one with MBP-tagged RMI1 and the other with Flag-tagged RMI1.…”

“…However, there are contrasting reports in the literature on the oligomeric state of BLM. Size exclusion chromatography and atomic force microscopy support monomeric, dimeric, and larger oligomeric states, while EM data reveal five- or six-lobed structures reminiscent of ring-shaped multimeric ATPases ( 24 – 27 ). The oligomeric state of BLM within the BS complex has never been investigated, although RMI1:RMI2 purified in isolation forms a 1:1 heterodimer in published reports ( 16 , 18 , 19 ).…”

A non-catalytic N-terminus domain of WRN prevents mitotic telomere deprotection

“…2). Amino acids 228-333 have been shown to contain a coiled-coil region required for multimerization of WRN 30,38 . The WRN fragments containing this domain showed a higher band shift on SDS-PAGE (Fig.…”

“…This band shift could be caused by resistance to SDS-induced denaturation, as reported in WRN fragments containing this domain 30 , although the band size does not agree with multimer sizes (Table S1). The multimerization domain of WRN has been linked to the processivity of exonuclease and efficient strand exchange activities 30,38 . In terms of activity, however, our results strongly argue that the mitotic effect of the N-terminus does not require the catalytic property of WRN.…”