Comparisons between the structures of HCV and Rep helicases reveal structural similarities between SF1 and SF2 super‐families of helicases

Abstract

Three helicase structures have been determined recently: that of the DNA helicase PcrA, that of the hepatitis C virus RNA helicase, and that of the Escherichia coli DNA helicase Rep. PcrA and Rep belong to the same super-family of helicases (SFI) and are structurally very similar. In contrast, the HCV helicase belongs to a different super-family of helicases, SF2, and shows little sequence homology with the PcrA/Rep helicases. Yet, the HCV helicase is structurally similar to Rep/PcrA, suggesting preservation of structural scaffolds and relationships between helicase motifs across these two super-families. The comparison study presented here also reveals the existence of a new helicase motif in the SF1 family of helicases.Keywords: HCV, helicase; helicase motif; Rep; structure Helicases are enzymes that unwind duplex DNA or RNA in reactions coupled to nucleoside 5' triphosphate (NTP) binding and hydrolysis, and play central roles in processes affecting nucleic acids (Lohman & Bjomson, 1996). On the basis of amino acid sequence homology, helicases have been classified into five superfamilies (Gorbalenya & Koonin, 1993). Although the overall sequence homology is low, conserved regions or "helicase" motifs within each family have been identified (Gorbalenya et al., 1989). However, homology across families is very weak and limited to the signature sequences that are required for NTP binding (helicase motifs I and 11) (Koonin & Dolja, 1993). With the exception of these NTP binding sequences, it has not been clear prior to the structural comparisons reported here whether the other motifs share common functional roles.Recently, structural information on three helicases has been reported at atomic resolution. The first view of a DNA-helicase, that of the PcrA helicase from Bacillus stearothemzophilus, revealed a two-domain structure (domains 1 and 2), with each domain consisting of two subdomains (lA, lB, 2A, and 2B) (Subramanya et al., 1996). While subdomains 1B and 2B are mostly a-helical and are formed by large insertions within subdomains 1A and 2A (Fig. lA) (Fig. IA). The second view of a helicase was that of the RNA helicase domain of the NS3 protein of hepatitis C virus (HCV) (Yao et al., 1997) (Fig. 1B). The HCV helicase consists of three distinct domains, two of which are RecA structural homologues. The third domain is mostly a-helical, and is formed by the C-terminal region of the sequence. Finally, a third view of a DNA helicase structure was recently obtained: that of the Rep DNA helicase bound to single-stranded DNA (ss-DNA) (Korolev et al., 1997). The asymmetric unit of the Rep/ss-DNA crystals contained two molecules that differ markedly by a large reorientation of the subdomain structure. Although one of the molecules (the open conformer) is very similar in structure to the PcrA DNA-helicase (Fig. lA), the other molecule (the closed form) has undergone a swiveling motion of its 2B domain, corresponding to a 130" rotation around the hinge region that connects domains 2A and 2B.In an attempt to deter...

Deletion of MUD2, the yeast homolog of U2AF65, can bypass the requirement for Sub2, an essential spliceosomal ATPase

“…1). Available crystal structures of superfamily 2 (SF2) helicases and related superfamily 1 (SF1) helicases place this motif in a structural domain implicated in RNA binding/helicase activity (Pause et al 1993;Korolev et al 1997Korolev et al , 1998Yao et al 1997;Bird et al 1998;Velankar et al 1999). In contrast, Figure 1.…”

“…These motifs have been shown to be required for ATP binding/hydrolysis and thus the coordination of conformational changes in the ATP binding domain with the function of the other motifs of SF2 helicases (Pause and Sonenberg 1992;Hotz and Schwer 1998;Edwalds-Gilbert et al 2000). Recent crystal structures of SF1 and SF2 helicases place motifs I and Ia in a structural domain distinct from that containing motif IV (Bird et al 1998;Korolev et al 1998;Johnson and McKay 1999). Thus, the differences in growth phenotypes of sub2-1 and sub2-5 probably reflect inactivation of different aspects of wild-type Sub2 function.…”

Defining the roles of individual residues in the single-stranded DNA binding site of PcrA helicase

“…F64 is located in motif Ia, and H587 is found in the region between the closely aligned motifs V and VI. F192 and F626 are outside of the signature motifs but are conserved within the Rep͞UvrD͞PcrA family of SF1 DNA helicases (15).…”

Velocity and processivity of helicase unwinding of double-stranded nucleic acids

“…A range of up to 10 bases is motivated by the typical size of a helicase monomer. Indeed, structures determined by X-ray diffraction suggest that a helicase can interact with 5-10 bases in the ds region of the ss-ds junction [47,48,49,50,51,52,53]. We find that for an active helicase the unwinding velocity can approach the single-strand translocation rate of the motor.…”