https://www.cell.com/cell/pdf/S0092-8674(09)01303-8.pdf

Concerted Loading of Mcm2-7 Double Hexamers Around DNA during DNA Replication Origin Licensing

During pre-replication complex (pre-RC) formation, origin recognition complex (ORC), Cdc6, and Cdt1 cooperatively load the 6-subunit mini chromosome maintenance (MCM2-7) complex onto DNA. Loading of MCM2-7 is a prerequisite for DNA licensing that restricts DNA replication to once per cell cycle. During S phase MCM2-7 functions as part of the replicative helicase but within the pre-RC MCM2-7 is inactive. The organization of replicative DNA helicases before and after loading onto DNA has been studied in bacteria and viruses but not eukaryotes and is of major importance for understanding the MCM2-7 loading mechanism and replisome assembly. Lack of an efficient reconstituted pre-RC system has hindered the detailed mechanistic and structural analysis of MCM2-7 loading for a long time. We have reconstituted Saccharomyces cerevisiae pre-RC formation with purified proteins and showed efficient loading of MCM2-7 onto origin DNA in vitro. MCM2-7 loading was found to be dependent on the presence of all pre-RC proteins, origin DNA, and ATP hydrolysis. The quaternary structure of MCM2-7 changes during pre-RC formation: MCM2-7 before loading is a single hexamer in solution but is transformed into a double-hexamer during pre-RC formation. Using electron microscopy (EM), we observed that loaded MCM2-7 encircles DNA. The loaded MCM2-7 complex can slide on DNA, and sliding is not directional. Our results provide key insights into mechanisms of pre-RC formation and have important implications for understanding the role of the MCM2-7 in establishment of bidirectional replication forks.

/pdf/a-double-hexameric-mcm2-7-complex-is-loaded-onto-origin-dna-1pjy1uta4d.pdf

A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication

The E1 protein of papillomavirus is a hexameric ring helicase belonging to the AAA + family. The mechanism that couples the ATP cycle to DNA translocation has been unclear. Here we present the crystal structure of the E1 hexamer with single-stranded DNA discretely bound within the hexamer channel and nucleotides at the subunit interfaces. This structure demonstrates that only one strand of DNA passes through the hexamer channel and that the DNA-binding hairpins of each subunit form a spiral 'staircase' that sequentially tracks the oligonucleotide backbone. Consecutively grouped ATP, ADP and apo configurations correlate with the height of the hairpin, suggesting a straightforward DNA translocation mechanism. Each subunit sequentially progresses through ATP, ADP and apo states while the associated DNA-binding hairpin travels from the top staircase position to the bottom, escorting one nucleotide of single-stranded DNA through the channel. These events permute sequentially around the ring from one subunit to the next.

Mechanism of DNA translocation in a replicative hexameric helicase

Summary: The Mcm2-7 complex serves as the eukaryotic replicative helicase, the molecular motor that both unwinds duplex DNA and powers fork progression during DNA replication. Consistent with its central role in this process, much prior work has illustrated that Mcm2-7 loading and activation are landmark events in the regulation of DNA replication. Unlike any other hexameric helicase, Mcm2-7 is composed of six unique and essential subunits. Although the unusual oligomeric nature of this complex has long hampered biochemical investigations, recent advances with both the eukaryotic as well as the simpler archaeal Mcm complexes provide mechanistic insight into their function. In contrast to better-studied homohexameric helicases, evidence suggests that the six Mcm2-7 complex ATPase active sites are functionally distinct and are likely specialized to accommodate the regulatory constraints of the eukaryotic process.

The Mcm Complex: Unwinding the Mechanism of a Replicative Helicase

The papillomavirus E2 proteins.

https://www.cell.com/molecular-cell/pdf/S1097-2765(05)01641-2.pdf

Assembly of a double hexameric helicase.

A prerequisite for DNA replication is the unwinding of duplex DNA catalyzed by a replicative hexameric helicase. Despite a growing body of research, key elements of helicase mechanism remain under substantial debate. In particular, the number of DNA strands encircled by the helicase ring during unwinding and the ring orientation at the replication fork completely contrast in contemporary mechanistic models. Here we use single-molecule and ensemble assays to address these questions for the papillomavirus E1 helicase. We find that E1 unwinds DNA with a strand-exclusion mechanism, with the N-terminal side of the helicase ring facing the replication fork. We show that E1 generates strikingly heterogeneous unwinding patterns stemming from varying degrees of repetitive movements, which is modulated by the DNA-binding domain. Together, our studies reveal previously unrecognized dynamic facets of replicative helicase unwinding mechanisms.

https://www.pnas.org/content/pnas/111/9/E827.full.pdf

Dynamic look at DNA unwinding by a replicative helicase

https://www.cell.com/molecular-cell/pdf/S1097-2765(07)00108-6.pdf

Adjacent Residues in the E1 Initiator β-Hairpin Define Different Roles of the β-Hairpin in Ori Melting, Helicase Loading, and Helicase Activity

Mechanistic Analysis of Local Ori Melting and Helicase Assembly by the Papillomavirus E1 Protein

Papillomaviruses have complex life cycles that are understood only superficially Although it is well established that the viral E1 and E2 proteins play key roles in controlling viral transcription and DNA replication, how these factors are regulated is not well understood Here, we demonstrate that phosphorylation by the protein kinase CK2 controls the biochemical activities of the bovine papillomavirus E1 and E2 proteins by modifying their DNA binding activity Phosphorylation at multiple sites in the N-terminal domain in E1 results in the loss of sequence-specific DNA binding activity, a feature that is also conserved in human papillomavirus (HPV) E1 proteins The bovine papillomavirus (BPV) E2 protein, when phosphorylated by CK2 on two specific sites in the hinge, also loses its site-specific DNA binding activity Mutation of these sites in E2 results in greatly increased levels of latent viral DNA replication, indicating that CK2 phosphorylation of E2 is a negative regulator of viral DNA replication during latent viral replication In contrast, mutation of the N-terminal phosphorylation sites in E1 has no effect on latent viral DNA replication We propose that the phosphorylation of the N terminus of E1 plays a role only in vegetative viral DNA replication, and consistent with such a role, caspase 3 cleavage of E1, which has been shown to be necessary for vegetative viral DNA replication, restores the DNA binding activity to phosphorylated E1

Stephen Schuck

Papers

Assembly of a double hexameric helicase.

Dynamic look at DNA unwinding by a replicative helicase

Adjacent Residues in the E1 Initiator β-Hairpin Define Different Roles of the β-Hairpin in Ori Melting, Helicase Loading, and Helicase Activity

Mechanistic Analysis of Local Ori Melting and Helicase Assembly by the Papillomavirus E1 Protein

CK2 Phosphorylation Inactivates DNA Binding by the Papillomavirus E1 and E2 Proteins