Two ReCA Domains Comprise a Minimal Functional Unit of the Human BlM Helicase

Abstract

Human Bloom's syndrome helicase (BLM), a member of the RecQ family, plays key roles in homologous recombination (HR) based DNA repair mechanisms. BLM is a multidomain protein comprising four major domains. i, The large N-terminal domain is responsible for oligomerization of the helicase, and for maintaining protein-protein interactions required for double Holliday-junction (DHJ) dissolution and D-loop disruption. ii, The core of BLM consists of two conserved RecA domains that form the ATP-binding cleft and bind unwound DNA segments. iii, The conserved RecQ-C-terminal domain (RQC) is located downstream of the RecA domains. This domain contains the Zn2+-binding region which is essential for correct folding. iv, The C-terminal HRDC (helicase- and RNase-D C-terminal) domain is indispensable for DHJ dissolution. In a quest for the minimal functional helicase core of BLM, we produced a series of truncated constructs and tested them for ssDNA binding and translocation, duplex unwinding, and clearance of hRad51 recombinase-nucleoprotein filaments. Surprisingly we found that the deletion of the N-terminal and HRDC domains (BLM642-1077) did not change the activity profile of BLM compared with the widely used RecQ-homolog construct (BLM642-1290), and the strand annealing activity was also retained. Moreover, a construct comprising only the two RecA domains (BLM642-1005), thus lacking even the RQC domain, unwound splayed-arm double stranded (ds) DNA substrate and displayed hRad51-clearance activity. However, this construct exhibited no ssDNA-dependent enzymatic activation, indicating no ssDNA translocation. In summary, these results suggest that the ‘helicase core’ of DEAH-box BLM works similarly to the DEAD-box RNA helicases (Vasa, eIF4A) in which the two RecA domains are capable of disrupting short dsRNA regions and nucleoprotein filaments with no processive translocation. In this sense, additional domains of BLM display modulatory partner-like functions increasing processivity and providing specific activities.