Coupling of DNA binding and helicase activity is mediated by a conserved loop in the MCM protein

Abstract

Minichromosome maintenance (MCM) helicases are the presumptive replicative helicases, thought to separate the two strands of chromosomal DNA during replication. In archaea, the catalytic activity resides within the C-terminal region of the MCM protein. In Methanothermobacter thermautotrophicus the N-terminal portion of the protein was shown to be involved in protein multimerization and binding to single and double stranded DNA. MCM homologues from many archaeal species have highly conserved predicted amino acid similarity in a loop located between β7 and β8 in the N-terminal part of the molecule. This high degree of conservation suggests a functional role for the loop. Mutational analysis and biochemical characterization of the conserved residues suggest that the loop participates in communication between the N-terminal portion of the helicase and the C-terminal catalytic domain. Since similar residues are also conserved in the eukaryotic MCM proteins, the data presented here suggest a similar coupling between the N-terminal and catalytic domain of the eukaryotic enzyme.

Crystal structure of a near-full-length archaeal MCM: Functional insights for an AAA+ hexameric helicase

“…S3C). These cis-and trans-N-C interactions revealed by the hexameric MCM model provide a molecular explanation for the observed cooperation of separately purified N and C domains, the reduced ATPase activity of the mtMCM R98 N-terminal mutation, and the variety of L207 mutations on the N domain that affect ATPase activity and helicase activity (19,22,25).…”

“…S3C). Mutational analysis of this loop has been recently performed (25). This loop was proposed to act as a medium for transmitting signals from ATP hydrolysis to the N-terminal domain.…”

“…Additionally, an R98A mutation in the mtMCM N domain (ssoMCM R110) reduces the DNA-stimulated ATPase activity of the C domain (19). Further, mutations on L207 by us and others (25) were shown to affect ATPase and helicase activity of ssoMCM and mtMCM. Our ssoMCM structure models reveal that the N and C domains interact with each other, not only within a single subunit (cis-N-C interactions, Fig.…”

The Mcm Complex: Unwinding the Mechanism of a Replicative Helicase

“…These charged residues appear to be important for DNA binding, as substitution mutations that neutralize these charges decrease DNA binding (79). With an affinity similar to those of other hexameric helicases (197), MthMcm binds ssDNA substrates in an Mg 2ϩ -dependent manner (K d for ssDNA of ϳ130 nM [42]), with a slight preference for ssDNA over dsDNA (213). The SsoMcm complex apparently prefers substrates that contain both ssDNA and dsDNA: forked DNA substrates were bound with higher affinity than partial dsDNA probes containing a poly(dT) bubble or a 5Ј ssDNA tail (62), but both forks and bubbles were preferred over simple ssDNA probes (204,209).…”

“…For SsoMcm, this domain has been biochemically studied in isolation, but the analogous region in MthMcm is insoluble and resists experimental analysis (213). In isolation, this domain is competent to bind DNA in an ATP-independent manner, hydrolyze ATP, and unwind DNA (13,203,213).…”