MCM ring hexamerization is a prerequisite for DNA-binding

Abstract

The hexameric Minichromosome Maintenance (MCM) protein complex forms a ring that unwinds DNA at the replication fork in eukaryotes and archaea. Our recent crystal structure of an archaeal MCM N-terminal domain bound to single-stranded DNA (ssDNA) revealed ssDNA associating across tight subunit interfaces but not at the loose interfaces, indicating that DNA-binding is governed not only by the DNA-binding residues of the subunits (MCM ssDNA-binding motif, MSSB) but also by the relative orientation of the subunits. We now extend these findings by showing that DNA-binding by the MCM N-terminal domain of the archaeal organism Pyrococcus furiosus occurs specifically in the hexameric oligomeric form. We show that mutants defective for hexamerization are defective in binding ssDNA despite retaining all the residues observed to interact with ssDNA in the crystal structure. One mutation that exhibits severely defective hexamerization and ssDNA-binding is at a conserved phenylalanine that aligns with the mouse Mcm4(Chaos3) mutation associated with chromosomal instability, cancer, and decreased intersubunit association.

Fundamental Characteristics of AAA+ Protein Family Structure and Function

“…The MCM complex is a hexameric ring helicase that separates parental DNA strands at the replication fork in eukaryotes and archaea. The X-ray crystal structures of archaeal MCM proteins have revealed overall architectural features that are highly conserved in both archaea and eukaryotes [ 3 , 41 , 81 – 85 ]. All MCM hexamers assemble with a two-tiered architecture with a larger C-terminal AAA+ tier and an N-terminal tier (Figures 3(a) and 3(d) ) [ 41 , 51 , 86 – 89 ].…”

Rac GTPase activating protein 1 promotes the glioma growth by regulating the expression of MCM3

“…MCM3, as an important component of the MCM2–7 complex, plays an important role in the cell cycle and DNA replication [38] . The hexameric proteins formed by MCM2-MCM7, which unwind DNA, are normally expressed during the cell cycle, and it has been reported that the MCM2–7 loop alone could unwinds double-stranded DNA in vitro in several archaea [ 39 , 40 ]. However, even a minor reduction in the MCM pool can negatively affect the integrity of the genome, as shown by DNA fragmentation, micronuclei formation, and genomic instability [41] .…”

DNA replication: Mechanisms and therapeutic interventions for diseases

“…The peripheral helical bundle interacts with the OB fraction via a slight linker promoting the interaction with DNA 58 . The observation indicated that a helical bundle might be essential for protein–protein interplay and protein–DNA interactions during the initiation step.…”

“…56,57 The peripheral helical bundle interacts with the OB fraction via a slight linker promoting the interaction with DNA. 58 The observation indicated that a helical bundle might be essential for protein-protein interplay and protein-DNA interactions during the initiation step. However, molecular mechanics studies presented that deleting a helical bundle exerts a limited influence on MCM function.…”