Mechanistic Basis of 5′-3′ Translocation in SF1B Helicases

Abstract

Superfamily 1B (SF1B) helicases translocate in a 5'-3' direction and are required for a range of cellular activities across all domains of life. However, structural analyses to date have focused on how SF1A helicases achieve 3'-5' movement along nucleic acids. We present crystal structures of the complex between the SF1B helicase RecD2 from Deinococcus radiodurans and ssDNA in the presence and absence of an ATP analog. These snapshots of the reaction pathway reveal a nucleotide binding-induced conformational change of the two motor domains that is broadly reminiscent of changes observed in other SF1 and SF2 helicases. Together with biochemical data, the structures point to a step size for translocation of one base per ATP hydrolyzed. Moreover, the structures also reveal a mechanism for nucleic acid translocation in the 5'-3' direction by SF1B helicases that is surprisingly different from that of 3'-5' translocation by SF1A enzymes, and explains the molecular basis of directionality.

Three conformational snapshots of the hepatitis C virus NS3 helicase reveal a ratchet translocation mechanism

“…3). Similar to NS3h, SF1 enzymes show closure of the two RecA-like domains upon ATP-analog binding (16)(17)(18). No intradomain 1 and 2 structural transitions analogous to those in HCV NS3h, however, have been observed.…”

“…For example, the base rotation around W501 (a potential "gatekeeper") probably allows NABS2 to slide and initiate the translocation, and DNA pucker alternations along with the backbone rotations may facilitate the motion. No analogous intradomain structural transitions have been observed in the SF1 enzymes, although ATP-induced rigid-body rotations of the motor domains are seen (16)(17)(18). Indeed, structural comparison of the representative SF1 and SF2 members reveals explicit differences in catalyzing nucleotide hydrolysis and motion (Figs.…”

“…Although crystal structures of SF2 helicases have been reported, no enzyme in complex with its substrate has been visualized in different conformational states and the structural details pertaining to the actions of these helicases are lacking (15,16). Despite the structural snapshots obtained for the SF1 helicases (16)(17)(18), the motif distinctions between SF1 and SF2 members suggest divergent mechanisms and there is no consensus on how the SF2 enzymes translocate unidirectionally along ssDNA/ssRNA and destabilize duplexes for unwinding (3,15). This deficiency has hampered efforts to understand the functional details of SF2 helicases and slowed the search for potent inhibitors targeting medically important candidates, such as the HCV helicase (3,19).…”

Erratum to: Structure and Mechanisms of SF1 DNA Helicases

“…Comparison of the structures of PcrA and RecD2 shows that both SF1A and SF1B helicases bind the ssDNA in the same orientation (2A domain on the 5 side of the DNA, 1A domain on the 3 side of the DNA) [89,94], and reveals how directionality is determined ( Fig. 2.5a, b) [89,94,127].…”

“…Some of the well-characterized SF1A helicases are PcrA, Rep, and UvrD T [87][88][89][90][91][92]. Well-characterized members of SF1B include RecD and Dda [93,94] (He et al, in press). The biological functions, polarity, and active forms of some of the SF1 helicases are listed (Table 2.2).…”