Mechanisms of hexameric helicases

Abstract

Ring-shaped hexameric helicases are essential motor proteins that separate duplex nucleic acid strands for DNA replication, recombination, and transcriptional regulation. Two evolutionarily distinct lineages of these enzymes, predicated on RecA and AAA+ ATPase folds, have been identified and characterized to date. Hexameric helicases couple NTP hydrolysis with conformational changes that move nucleic acid substrates through a central pore in the enzyme. How hexameric helicases productively engage client DNA or RNA segments and use successive rounds of NTPase activity to power translocation and unwinding have been longstanding questions in the field. Recent structural and biophysical findings are beginning to reveal commonalities in NTP hydrolysis and substrate translocation by diverse hexameric helicase families. Here, we review these molecular mechanisms and highlight aspects of their function that are yet to be understood.

Keywords: AAA+; ATPase; DNA replication; DnaB; Helicase; MCM; RecA; Rho.

Fig. 1:. Hexameric helicase superfamilies and folds.

The core ASCE αβα binding fold (blue) has diverged into the RecA (pink) and AAA+ (green) families that make up all hexameric helicases. Select structural insertions and locations or specific motifs are indicated (yellow for ASCE, pink or green for RecA and AAA+, respectively). Dark green dotted lines and arrows show the helix two insert (H2I) that is specific to SF-VI and not found in SF-III helicases.

Fig. 2:. Overview of RecA and AAA+ hexameric helicase structures.

A. SF-IV – G. stearothermophilus DnaB bound to ssDNA and GDP•AlF₄ (PDB 4ESV). B. SF-V – E. coli Rho bound to ADP•BeF₃ and RNA (PDB 3ICE). C. SF-III – papillomavirus E1 bound to ADP and ssDNA (PDB 2GXA). D. SF-VI – D. melanogaster Mcm2-7 bound to ADP, ATP and ssDNA (PDB 6RAZ). For all – Top: Top-down view of hexameric rings and active sites (inset). Bottom: Side view showing N-and C-terminal domains. RF = Arginine Finger, WA = Walker A, WB = Walker B, CE = Catalytic Glutamate, SI = Sensor I, SII = Sensor II.

Fig. 3:. Nucleic acid binding and ATPase status of hexameric helicases.

A. SF-IV – G. stearothermophilus DnaB. All GTP-bound active sites are in the same conformation and thus ATPase status around the ring cannot be assigned (PDB 4ESV). B. SF-V – E. coli Rho with ATPase state assigned by active site conformation and nucleotide status (PDB 3ICE). C. SF-III – papillomavirus E1 with ATPase state assigned by active site conformation (PDB 2GXA). D. SF-VI – D. melanogaster Mcm2-7 with ATPase state assigned by cryo-EM density and active site conformation (PDB 6RAZ). For all – nucleic acid substrates are shown in blue and pore loops in red. Nucleotide states: ATP – substrate bound; ADP – product bound; E – empty/exchangeable, ATP* - hydrolysis competent. All views are looking down from the 3’-5’ perspective. Insets show cutaway of helicase structure to highlight pore loops interactions with nucleic acid substrates.

Fig. 4:. Models for NTP hydrolysis.

ATPase states: T (green) – ATP; D (orange) – ADP + P_i; E (red) – empty. A. Concerted hydrolysis. All sites fire in synchrony. B. Stochastic hydrolysis. Sites fire at random. C. Rotary hydrolysis. Sites fire in a sequential order around the ring.

Fig. 5:. Models for translocation.

A. Pumpjack translocation where a stable N-terminal region (gray) encircles ssDNA while the C-terminal motor regions moves up and down as a levering unit. B. Hand-over-hand translocation in which rounds of ATP binding, hydrolysis, and product release coincide with an entire subunit moving from one end of the helicase spiral to the other (bottom-to-top movement as depicted here). C. Concerted escort translocation in which DNA binding loops (red, inset) form a spiral around DNA and rounds of ATP binding, hydrolysis, and product release are coupled to upward and downward loop movements (bottom-to-top movement as depicted here). Red arrows indicate the direction of translocation.

Fig. 6:. Power stroke vs. Brownian ratchet unwinding mechanisms.

A. In the power stroke model, ATP hydrolysis by the helicase is tied to conformational changes that actively push it forward into a duplex, forcibly splitting paired bases to unwind the substrate. B. Passive unwinding involves local fraying of base pairs due to thermal or torsional fluctuations that expose ssDNA, which the helicase then captures through an ATP-dependent movement.

Fig. 7:. Strategies for loading hexameric helicases.

A. Ring assembly. SF-III helicases such as LTag and E1 are assembled at viral replication origins, proceeding through head-to-head intermediate complexes into single hexamers that encircle single DNA strands. B. Chaperoned rings opening. The SF-IV DnaB helicase from E. coli forms pre-assembled hexamers that are physically opened by DnaC and loaded onto a ssDNA bubble formed by the replication initiator, DnaA. C. Self-regulated ring closure. The SF-V helicase Rho forms a pre-opened hexamer that is recruited to target RNAs by Rho utilization (rut) sequences; once bound, the threading of ssRNA into the helicase channel promotes ring closure. D. Chaperoned ring closure. SF-VI helicases like MCMs form pre-opened hexameric rings that are loaded onto duplex replication origins by ORC, Cdc6, and Cdt1. This process establishes a closed-ring, head-to-head double hexamer intermediate that, when activated by Cdc45 and GINS to form the CMG complex, melts the origin and isomerizes into single hexamers that encircle ssDNA. Red arrows indicate direction of post-loading translocation. Helicase bypass following loading occurs for SF-III, -IV, and -VI enzymes.