A double-hexamer archaeal minichromosome maintenance protein is an ATP-dependent DNA helicase

Abstract

The minichromosome maintenance (MCM) proteins are essential for DNA replication in eukaryotes. Thus far, all eukaryotes have been shown to contain six highly related MCMs that apparently function together in DNA replication. Sequencing of the entire genome of the thermophilic archaeon Methanobacterium thermoautotrophicum has allowed us to identify only a single MCM-like gene (ORF Mt1770). This gene is most similar to MCM4 in eukaryotic cells. Here we have expressed and purified the M. thermoautotrophicum MCM protein. The purified protein forms a complex that has a molecular mass of approximately 850 kDa, consistent with formation of a double hexamer. The protein has an ATP-independent DNA-binding activity, a DNA-stimulated ATPase activity that discriminates between single- and double-stranded DNA, and a strand-displacement (helicase) activity that can unwind up to 500 base pairs. The 3' to 5' helicase activity requires both ATP hydrolysis and a functional nucleotide-binding site. Moreover, the double hexamer form is the active helicase. It is therefore likely that an MCM complex acts as the replicative DNA helicase in eukaryotes and archaea. The simplified replication machinery in archaea may provide a simplified model for assembly of the machinery required for initiation of eukaryotic DNA replication.

Figure 1

MtMCM protein shows DNA-stimulated ATPase activity. (A) ≈360 fmol of 12-mer MtMCM was assayed for ATPase activity in the presence of 90, 180, 360, 720, and 1,440 fmol (lanes 4–8, 12–16) and 2,880 fmol (lanes 9, 10 and 17, 18) of closed circular pUC118 ssDNA or pUC118, respectively. (B) ATPase activity was plotted against the concentration of closed circular single-stranded pUC118 (○) or double-stranded pUC118 (■). (C) The quantified data were replotted as a function of the molar ratio of [protein]/[DNA] (using the same convention as for B). For the purpose of the calculation, MtMCM was assumed to form double hexamers.

Figure 2

MtMCM requires Mg²⁺ but not ATP to bind DNA. One hundred nanograms (lanes 2, 10, 18), 200 ng (lanes 3, 11, 19), 400 ng (lanes 4, 12, 20), and 800 ng (lanes 5–9, 13–17, 21–25) of wt and mutant protein were incubated with 20 fmol of labeled oligomer in the presence of 4 mM ATP (lanes 2–6, 10–14, 18–22), 4 mM γ-thio-ATP (ATP[γ-S]; lanes 8, 18, 24), 4 mM 5′-[β,γ-imido]triphosphate (AMP-PNP; lanes 9, 17, 25). Lanes 7, 15, and 23 did not contain ATP, and lanes 6, 14, and 22 were supplemented with an additional 20 mM EDTA to chelate free metal ions.

Figure 3

MtMCM shows an ATP-dependent DNA helicase activity. (A) Reaction mixtures (20 μl) containing 100 ng (lanes 3, 11, 15), 200 ng (lanes 4, 12, 16), 400 ng (lanes 5, 13, 17), or 800 ng (lanes 6–10, 14, 18) of protein were incubated with ≈10 fmol of substrate and 4 mM ATP (lanes 3–7, 11–18), 4 mM ATP[γ-S] (lane 9), 4 mM 5′-[β,γ-imido]triphosphate (AMP-PNP; lane 10), or 20 mM EDTA (lane 7). (B) Oligonucleotides labeled at the 5′ or 3′ ends were annealed to pUC118 ssDNA and digested to produce linear substrates that were used to measure polarity of displacement. (C) MtMCM protein (100, 200, 400, and 800 ng) was incubated with ≈300 fmol of linear substrate in helicase assays. The displaced oligonucleotide was compared with an equal amount of heat-denatured substrate. Displaced 5′-labeled (○) and 3′-labeled (●) oligonucleotides were quantified on a phosphorimager.

Figure 4

wt MtMCM is capable of displacing up to 500 bp of DNA without additional proteins. ≈10 fmol of long substrate was incubated with 100 ng (lanes 5, 10, 15), 200 ng (lanes 6, 11, 16), 400 ng (lanes 7, 12, 17), or 800 ng (lanes 8, 9, 13, 14, 18, 19) of protein for 30 min at 37°C. Displaced oligonucleotides were separated by PAGE.

Figure 5

Estimation of the native molecular mass of MtMCM protein. (A) wt and ΔN proteins were sized by sucrose gradient sedimentation, and fractions were subjected to SDS/PAGE and stained with Coomassie blue. (B) Samples were also sized by gel filtration. Gel filtration fractions were assayed for ATPase activity (■). Activity coeluted only with the peak of wt protein.

Figure 6

wt MtMCM protein forms a double hexamer. (A) Samples of wt protein were subjected to scanning transmission electron microscopy. Separate mass estimates of end (arrowhead) and side views (arrow) were based on unstained sample measurements. (B) Stained samples showed a central pore in the end view and suggested that the complexes consisted of a double hexamer. (Bar = 20 nm.)