The effects of oligomerization on Saccharomyces cerevisiae Mcm4/6/7 function

Abstract

BackgroundMinichromosome maintenance proteins (Mcm) 2, 3, 4, 5, 6 and 7 are related by sequence and form a variety of complexes that unwind DNA, including Mcm4/6/7. A Mcm4/6/7 trimer forms one half of the Mcm2-7 hexameric ring and can be thought of as the catalytic core of Mcm2-7, the replicative helicase in eukaryotic cells. Oligomeric analysis of Mcm4/6/7 suggests that it forms a hexamer containing two Mcm4/6/7 trimers, however, under certain conditions trimeric Mcm4/6/7 has also been observed. The functional significance of the different Mcm4/6/7 oligomeric states has not been assessed. The results of such an assessment would have implications for studies of both Mcm4/6/7 and Mcm2-7.ResultsHere, we show that Saccharomyces cerevisiae Mcm4/6/7 reconstituted from individual subunits exists in an equilibrium of oligomeric forms in which smaller oligomers predominate in the absence of ATP. In addition, we found that ATP, which is required for Mcm4/6/7 activity, shifts the equilibrium towards larger oligomers, likely hexamers of Mcm4/6/7. ATPγS and to a lesser extent ADP also shift the equilibrium towards hexamers. Study of Mcm4/6/7 complexes containing mutations that interfere with the formation of inter-subunit ATP sites (arginine finger mutants) indicates that full activity of Mcm4/6/7 requires all of its ATP sites, which are formed in a hexamer and not a trimer. In keeping with this observation, Mcm4/6/7 binds DNA as a hexamer.ConclusionsThe minimal functional unit of Mcm4/6/7 is a hexamer. One of the roles of ATP binding by Mcm4/6/7 may be to stabilize formation of hexamers.

Expression, purification and biochemical characterization of Schizosaccharomyces pombe Mcm4, 6 and 7

“…Our data support the arrangement model of the Mcm4/6/7 hexamer for the six subunits of spMCM (Figure 7B) that was proposed for S. cerevisiae MCM [26], and human MCM [23,24,42]. An alternative arrangement model, in which binding between Mcm6 and Mcm7 occurs, was proposed in a previous report [43]. The literatures have some disagreement about the interactions between Mcm6 and Mcm7.…”

Efficient expression and purification of human replication fork-stabilizing factor, Claspin, from mammalian cells: DNA-binding activity and novel protein interactions

“…3C). In order to evaluate the size of Claspin more precisely, Claspin was treated with glutaraldehyde (Ma et al 2010) and analyzed on a native gel and SDS-PAGE (Fig. 3C).…”

DNA replication: Mechanisms and therapeutic interventions for diseases

“…67 MCM4/6/7 proteins exhibit distinct functions when the ATP binding sites undergo mutations. 68 It should also be mentioned that MCM7 is required for ATP hydrolysis and DNA helicase via its ATP binding motif. 69 Nuclear magnetic resonance (NMR) structure studies revealed that the C-terminus of MCMs comprises a winged helix (WH) domain.…”

“… 67 MCM4/6/7 proteins exhibit distinct functions when the ATP binding sites undergo mutations. 68 It should also be mentioned that MCM7 is required for ATP hydrolysis and DNA helicase via its ATP binding motif. 69 …”

“…Despite all the MCMs harboring ATP‐binding motifs at the intersubunit interfaces, the ATP‐binding mode is quite different 67 . MCM4/6/7 proteins exhibit distinct functions when the ATP binding sites undergo mutations 68 . It should also be mentioned that MCM7 is required for ATP hydrolysis and DNA helicase via its ATP binding motif 69 …”