Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding

Abstract

Background: The mini-chromosome maintenance protein (MCM) complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7), the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM), six subunits of which form a homohexamer. We have recently reported a 4.35A crystal structure of the near full-length ssoMCM. The structure reveals a total of four beta-hairpins per subunit, three of which are located within the main channel or side channels of the ssoMCM hexamer model generated based on the symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM) structure. The fourth beta-hairpin, however, is located on the exterior of the hexamer, near the exit of the putative side channels and next to the ATP binding pocket.

Results: In order to better understand this hairpin's role in DNA binding and helicase activity, we performed a detailed mutational and biochemical analysis of nine residues on this exterior beta-hairpin (EXT-hp). We examined the activities of the mutants related to their helicase function, including hexamerization, ATPase, DNA binding and helicase activities. The assays showed that some of the residues on this EXT-hp play a role for DNA binding as well as for helicase activity.

Conclusions: These results implicate several current theories regarding helicase activity by this critical hexameric enzyme. As the data suggest that EXT-hp is involved in DNA binding, the results reported here imply that the EXT-hp located near the exterior exit of the side channels may play a role in contacting DNA substrate in a manner that affects DNA unwinding.

Figure 1

Overview of EXT-hp mutations. A) Sequence alignments of MCMs across archaea (top) and eukaryotes (bottom), for the short region corresponding to the EXT-hp. Sso: Sulfolobus solfataricus. Sac: Sulfolobus acidocaldarius. Prn: Aeropyrum pernix. Mth: Methanothermobacter thermautotrophicus. Pyf: Pyrococcus furiosus. Tap: Thermoplasma acidophilum. Sp: Schizosaccharomyces pombe. Sc: Saccharomyces cerevisiae. Hs: Homo sapiens. Xl: Xenopus laevis. Dm: Drosophila melanogaster. At: Arabidopsis thaliana. B) Table of mutations used for this work. C) Tilted side view of the ssoMCM hexamer model, showing the EXT-hp on the external side of the hexamer and near the side channel. A single subunit is shown in cyan, with its EXT-hp in red. The ATP binding pocket and side-channel are indicated. D) Close up view of EXT-hp. Inside and outside refer to towards the central channel and away from the central channel, respectively. The locations of the residues mutated in this study are colored on the EXT-hp according to amino acid type, as in panel B.

Figure 2

The results of FPLC and ATPase analysis. A) FPLC analysis of the mutations by gel filtration chromatography on a Superose-6 column. Molecular marker positions, Ferritin (440 kD) and aldolase (158 kD), are indicated by arrows. B) SDS-PAGE gel analysis of the purified mutant proteins. C) ATPase activity curves for WT, M3 and M8 in the presence and absence of Y-shaped DNA. Error bars representing standard error of the mean are present, but in most cases are too small to see. D) Summary of ATPase data in the presence (black bar) and absence (white bar) of Y-shaped DNA. Error bars are standard error from curve fitting.

Figure 3

Results of DNA binding assays. A) Representative EMSA gels assaying for DNA binding for WT and M8. Black triangle indicates increasing protein concentration. Locations of DNA alone and DNA-protein complexes are indicated. B) The curves of DNA binding for WT and M8. Error bars represent the standard error of the mean. C) Summary of DNA binding data for all mutants. Error bars represent standard error from curve fitting.

Figure 4

The results of helicase assays. A) Representative gel analysis result of helicase assay. B: boiled. UB: unboiled. DNA positions for Y-DNA and ssDNA are indicated. B) Summary of the quantified helicase activity of all mutants. Error bars represent standard error of the mean.