We have elucidated the mechanism of DNA translocation in a replicative hexameric helicase based on our structure of such a helicase with single-stranded DNA discretely bound within the hexamer channel and nucleotides at the subunit interfaces. We elaborated on this mechanism subsequently using single-molecule FRET studies. Based on structures of different oligomeric forms of the DNA-binding domain of the helicase with double-stranded DNA, we suggested a mechanism for strand separation.
The different faces of E1 - a replicative hexameric helicase
Precise replication of the genome is essential for maintaining the integrity of genomic information. As a prerequisite for DNA replication, two complementary DNA strands are separated and each becomes a template for the synthesis of a new complementary strand. Strand separation is mediated by a helicase enzyme, a molecular machine that uses the energy derived from ATP-hydrolysis while moving along the DNA. Our crystal structures of the dsDNA binding domain (DBD) of the replicative helicase E1 from papillomavirus in various stages of assembly, led us to propose a model in which the transition from the dimer to the ultimate double hexamer, results in strand separation. The loading and assembly of this protein separates the double helix, such that each hexameric helicase encircles one strand of DNA. Once assembled, the helicase uses its ATP-driven motor to translocate on the DNA or “pump” the ssDNA through the hexameric ring. Several competing mechanisms for helicase unwinding were proposed. Having determined a structure of hexameric E1 with ssDNA discretely bound in the central channel and nucleotides at the subunit interfaces, we showed that only one DNA strand passes through the hexamer channel and that the DNA-binding hairpins of each subunit form a spiral staircase that sequentially tracks the DNA backbone. The nucleotide configurations at the subunit interfaces indicate that each subunit sequentially progresses through ATP, ADP and apo states, while its associated DNA-binding hairpin travels from the top to the bottom of the staircase, each escorting one nucleotide of ssDNA through the channel, as if six hands grab the DNA and upon ATP hydrolysis and ADP release pull it through the channel. With this unique look into the mechanism of translocation of this molecular machine along DNA, we have focused on mechanistic aspects of the enzyme in solution.
By taking a multi-faceted approach including single-molecule and ensemble FRET (Förster Resonance Energy Transfer) methods, we have found that E1 is oriented with the N-terminal side of helicase facing the replication fork, consistent with the crystal structure. We also showed that E1 generates strikingly heterogeneous unwinding patterns stemming from varying degrees of repetitive movements that are modulated by the DNA-binding domain. Furthermore, our studies found that DNA-binding domain promotes the assembly of E1 helicase onto a forked DNA substrate, acting as an allosteric effector of the helicase. Our studies reveal previously unrecognized dynamic facets of replicative helicase unwinding mechanisms, adding another layer of complexity in the workings and regulation of DNA replication.