Dynamic coupling between the motors of DNA replication: hexameric helicase, DNA polymerase, and primase

Abstract

Helicases are molecular motor proteins that couple NTP hydrolysis to directional movement along nucleic acids. A class of helicases characterized by their ring-shaped hexameric structures translocate processively and unidirectionally along single-stranded (ss) DNA to separate the strands of double-stranded (ds) DNA, aiding both in the initiation and fork progression during DNA replication. These replicative ring-shaped helicases are found from virus to human. We review recent biochemical and structural studies that have expanded our understanding on how hexameric helicases use the NTPase reaction to translocate on ssDNA, unwind dsDNA, and how their physical and functional interactions with the DNA polymerase and primase enzymes coordinate replication of the two strands of dsDNA.

Figure 1. Structure of RecA family and AAA+ family hexameric helicases

The N-terminal end view of (a) Papillomavirus E1 hexameric helicase (PDB ID: 2GXA) and (b) Rho hexamer (PDB ID: 3ICE). Oligomerization domain of E1 (amino acids 308–378) and the N terminal domain of Rho (amino acids 1–130) are shown in blue. Nucleic acid (DNA for E1 and RNA for Rho) is bound in the central channel of the helicase with its 5’-end oriented towards the N-terminal domain. Each hexamer has 6 nucleotide binding sites at the subunit interfaces. The bound nucleotides (ADP BeF₃ in Rho and ADP in E1) are in pink. (c) Side views of E1 (left) and Rho (right). The nucleic acid (in spheres) is in the central channel and its orientation is indicated by the black arrow and Direction of translocation by the thick blue arrows.

Figure 2. Sequential mechanism of NTP hydrolysis and translocation on single stranded nucleic acid by Rho helicase

(a) The ss nucleic acid (blue shaded color) is held in place by the DNA binding loops (brown color) projecting out of the hexamer subunits inside the central channel. Five subunits interact with five nucleotides of ss nucleic acid in a spiral staircase fashion. Darker color of subunits and thicker loops indicate tighter interaction with NTP and nucleic acid. (b) Schematic representation of the NTP-ligated states of the subunits in the hexamer during translocation along ss nucleic acid. The solid black arrows indicate the direction of the sequential stages of NTP hydrolysis. In the sequential mechanism shown, the E subunit reels in a nucleotide of the nucleic acid (indicated by solid blue arrow) upon binding a new NTP. At the same time, the D subunit releases a nucleotide of the nucleic acid upon releasing NDP, the T subunit hydrolyzes NTP, and the DP* releases Pi.

Figure 3. Motors of the replisome: dynamics and interactions

The replisome is a complex dynamic molecular system which involves a variety of proteins functioning interdependently to ensure efficient synthesis of both leading and lagging strands. A model system of the proteins at the replication fork is depicted using the crystal structures of T7 helicase-primase gp4 (PDB ID: 1Q57) and DNA polymerase gp5 with its processivity factor thioredoxin (PDB ID: 1T7P). The helicase domain is shown in green, primase domain in red, DNA polymerase in purple and thioredoxin in light blue. The 3 ^rd polymerase proposed by recent studies to be a part of the replisome potentially to increase DNA synthesis processivity or bypass/repair lesions in the DNA, is shown in gray. (b) The rates of the individual motor proteins functioning by themselves or in a complex with other proteins of the replisome.

Figure 4. Coupled and uncoupled primosome in the replication machinery

(a) In the coupled primosome, the primase (in red) is making primer while it is engaged with the leading strand helicase-polymerase. The primase and primer remain bound to the forward moving leading strand replisome until handoff to the lagging polymerase; hence, the nascent ssDNA is looped out into a priming loop. (b) In the uncoupled primosome, the primase dissociates from the leading strand helicase-polymerase after primer synthesis.