Abstract
Helicases are ubiquitous enzymes that are involved in all kinds of DNA/RNA metabolisms including transcription, recombination and replication. Although the most basic function of most helicases is to unwind a double stranded nucleic acid substrate, their properties vary a lot from one helicase family to another. Sometimes even within the same subfamily, the mechanistic properties of two helicases can be very different. S. cerevisiae Upf1 (yUpf1) and human Smubp-2 are two helicases from Upf1-like family that belong to SF1B. In this work we show that despite having a similar helicase core, the helicase domain (HD) of these two enzymes work in a very different manner. yUpf1-HD has a strong binding affinity to ssDNA and a very high processivity, whereas Smubp-2-HD has week binding to ssDNA and almost unrecognizable processivity. Helicase cores of Smubp2 and Upf1 have similar folds: they contain two RecA-like domains (1A and 2A) and two sub-domains, 1B and 1C, inserted in domain 1A. Rec-A like domains are believed to act as a motor in a helicase. Hence it is puzzling why the two helicases behave so differently despite having highly conserved domains 1A and 2A. We try to identify the role of sub-domains 1B and 1C in this respect. We have utilized a magnetic tweezers based single molecule assay, acting in parallel on tens of molecules at the same time, which allows detecting the unwinding of DNA hairpin substrates. Using this system, we have studied various chimeras of yUpf1-HD and Smubp-2-HD. We show that domains 1B and 1C play an equally important role in processivity of these helicases.
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