Minichromosome maintenance helicase activity is controlled by N- and C-terminal motifs and requires the ATPase domain helix-2 insert

Abstract

The minichromosome maintenance (MCM) proteins are essential conserved proteins required for DNA replication in archaea and eukaryotes. MCM proteins are believed to provide the replicative helicase activity that unwinds template DNA ahead of the replication fork. Consistent with this hypothesis, MCM proteins can form hexameric complexes that possess ATP-dependent DNA unwinding activity. The molecular mechanism by which the energy of ATP hydrolysis is harnessed to DNA unwinding is unknown, although the ATPase activity has been attributed to a highly conserved AAA+ family ATPase domain. Here we show that changes to N- and C-terminal motifs in the single MCM protein from the archaeon Methanothermobacter thermautotrophicus (MthMCM) can modulate ATP hydrolysis, DNA binding, and duplex unwinding. Furthermore, these motifs appear to influence the movement of the β-α-β insert in helix-2 of the MCM ATPase domain. Removal of this motif from MthMCM increased dsDNA-stimulated ATP hydrolysis and increased the affinity of the mutant complex for ssDNA and dsDNA. Deletion of the helix-2 insert additionally resulted in the abrogation of DNA unwinding. Our results provide significant insight into the molecular mechanisms used by the MCM helicase to both regulate and execute DNA unwinding.

The minichromosome maintenance (MCM) proteins are conserved throughout eukaryotes and are essential for DNA replication initiation (1, 2) and progression (2). All eukaryotic organisms possess six highly related MCM proteins (MCM2–7) (3) that form high-molecular-weight complexes consistent with heterohexamers. Although homohexameric DNA helicases are relatively common and include the well studied SV40 large T antigen, papillomavirus E1, and phage T4 gene 4 protein, the MCMs are unique in forming a heterohexameric helicase. The evolutionary advantages presented by the additional complexity of a heterohexameric complex are not clear but may include specialization of individual components to allow interaction with a range of protein partners, allowing the replicative helicase to respond to a variety of cellular conditions. For example, recent evidence suggests that, in addition to DNA replication, MCMs play a role in transcriptional activation (4) and DNA damage checkpoint progression (5, 6).

The primary sequence of MCM proteins can be divided into three domains (7, 8): an N-terminal domain possessing a zinc-finger motif, a central AAA+ ATPase domain, and a C-terminal domain of unknown function (Fig. 5a). Consistent with this, eukaryotic MCM complexes have been shown to possess ATPase activity (9–12). In addition to the Walker A and B motifs of the ATPase domain, an arginine finger is required for ATP hydrolysis (9).

Fig. 5.

Identification and modeling of the h-2i in MthMCM. (a) Domain structure of MCMs, showing zinc-finger containing N-terminal domain, central AAA+ ATPase domain containing Walker A and B motifs, and h-2i. (b) Excerpt from clustalx alignment of the MCM ATPase domain showing the Walker A and B motifs (orange), the conserved presensor 1 β-hairpin lysine (blue), and the β-α-β h-2i (magenta). MCM sequences are Mth, M. thermautotrophicus; Sso, Sulfolobus solfataricus; Sc, Saccharomyces cerevisiae; and Hs, Homo sapiens. Consensus residues are shown in gray and are based on 100% agreement with the Betts and Russell scheme (33): ∗, identical; +, favorable substitution; −, neutral substitution. Side (c) and top (d) views of a molecular model of the N-terminal and ATPase domains of MthMCM. Walker motifs (orange) and h-2i (magenta) are highlighted in the magnesium chelatase structure (green). N-terminal crystal structure of MCM (magenta). Electron density mesh indicates overall complex conformation.

Although much evidence suggests that heterohexameric complexes of all six MCM proteins form (9, 11, 13, 14) and all six MCM proteins are required for DNA replication progression (2), to date there is no biochemical evidence of such a complex possessing processive helicase activity. An MCM 4,6,7 subcomplex possesses weak helicase activity in vitro (10, 11) that is improved in the presence of forked DNA substrates and ssDNA-binding protein (15). It is possible that in vivo MCM complex processivity is further improved by posttranslational modifications (e.g., ref. 16) or the presence of additional processivity factors.

The complexity of the eukaryotic MCM system means that addressing the molecular mechanisms required for unwinding has been difficult. The archaea also possess MCM proteins but in a simplified form. Almost all archaeal species possess a single MCM that forms homohexameric complexes. Archaeal MCMs are useful models for understanding some of the molecular mechanisms underlying the eukaryotic heterohexameric MCM complex (17). Biochemical studies of the Methanothermobacter thermautotrophicus MCM (MthMCM) demonstrated that this protein forms double-hexamer complexes that bind ssDNA and dsDNA. MthMCM also possesses dsDNA-stimulated ATPase and ATP-dependent DNA unwinding activities (18–20). A crystal structure of the N-terminal domain of MthMCM showed the subunits form a dodecameric ring-shaped complex with a central channel large enough to accommodate dsDNA (21). Hexameric, heptameric, and double hexameric complexes have been observed by 3D reconstruction of the full-length protein from electron microscope images (8, 22, 23).

MCMs possess two β-hairpins, one located in the N-terminal domain, which has a role in DNA binding (21, 24), and the presensor 1 β-hairpin in the ATPase domain (PS1BH), which is required for DNA duplex unwinding (24). The PS1BH is characteristic of superfamily 3 helicases and has been observed to move up to 17 Å during ATP binding and hydrolysis (25). This ATP hydrolysis-coupled movement is believed to cause translocation of DNA relative to the helicase complex; a point mutation of the PS1BH that would prevent DNA binding caused the abrogation of helicase activity in the Sulfolobus MCM (24).

Here we investigate the role of N- and C-terminal residues on MCM activity. We show that amino acids at both ends of the protein play roles in the modulation of ATP hydrolysis, DNA binding, and processivity of DNA unwinding. Changes to N- and C-terminal motifs can be related to hydrophobicity changes consistent with conformational changes in the ATPase domain of the protein. Finally, we demonstrate the key role of an ATPase domain β-α-β insert in transducing the energy of ATP hydrolysis into DNA unwinding. Among the superfamily 3 helicases, this motif is found uniquely in the MCMs. Together, our results suggest that ATP hydrolysis is optimized for DNA unwinding in the MCMs, and that helicase activity is carefully controlled.

Results

Generation of N- and C-Terminal Mutations.

A series of N-terminal MthMCM deletion mutants were generated by site-directed mutagenesis, purified, and tested for dsDNA-stimulated ATPase activity (not shown). We observed that the MthMCM complex lost ATPase activity between residues 89 and 105. An alignment of MCM sequences from a range of species identified a hydrophobic patch containing a single conserved arginine (R98) that we mutated to alanine to produce MthMCM R98A. Limited proteolysis of the full-length protein allowed us to identify a cleavable C-terminal domain. We recreated this truncated protein (Δ597) by the introduction of a stop codon into the expression vector. The R98A and Δ597 mutations were combined to produce a third mutant construct designated RAΔC. WT MthMCM, R98A, Δ597, and RAΔC proteins were expressed and purified. The mutant proteins all formed stable high-molecular-weight complexes consistent with double hexamers, suggesting that neither the R98 nor the C-terminal 72 amino acids play any significant role in the assembly or maintenance of multimeric MthMCM complexes.

ATPase Activity of MthMCM Mutants.

The WT and mutant proteins were subjected to ATPase assays in the presence of single- or double-stranded closed circular DNA (Fig. 1). As previously reported, the WT protein showed dsDNA-stimulated ATPase activity. Compared with WT, in the presence of dsDNA, the R98A mutant showed reduced levels of ATPase activity, whereas the Δ597 mutant showed an ≈3-fold increase in ATP hydrolysis. In the presence of dsDNA, RAΔC showed low levels of ATP hydrolysis that were above background. ATPase activity of RAΔC in the presence of ssDNA was identical to background in the absence of DNA (Fig. 1d Inset). We conclude that residues in both the N- and C-terminal domains of MthMCM can influence dsDNA-stimulated ATPase activity.

Fig. 1.

ATPase activity is enhanced in Δ597 but reduced in other MthMCM mutants. Quantification of ATP hydrolysis (calibrated from a standard curve produced by substrate dilution) for WT (a), R98A (b), Δ597 (c), and RAΔC (d) in the presence of dsDNA (closed circles, solid lines) or ssDNA (open circles, dotted lines). ATP hydrolysis in the absence of DNA is shown for RAΔC (d Inset, open squares).

DNA-Binding Activity of MthMCM Mutants.

We tested the DNA-binding activity of the MCM mutants using an EMSA. Both single- and double-stranded short linear DNA templates were tested in the presence and absence of ATP. Table 1 lists dissociation constants (K_d) for substrate saturation by WT and mutant proteins determined from the data in Fig. 7, which is published as supporting information on the PNAS web site. The MthMCM complex shows a preference for binding ssDNA rather than dsDNA. Consistent with previous results (18), DNA binding by the WT MthMCM was reduced in the presence of hydrolyzable ATP. All of the mutants showed approximately WT levels of dsDNA binding in the presence of ATP (Fig. 7). The R98A protein showed reduced affinity for ssDNA, whereas the Δ597 protein showed a higher than WT affinity for ssDNA and dsDNA under these conditions. Our results clearly indicate that the presence of hydrolyzable ATP reduces the affinity of MthMCM for DNA substrates. Three obvious mechanisms could explain these observations: (i) the presence of ATP could cause translocation of the DNA through the MCM complex followed by substrate release, (ii) ATP and DNA could compete for a particular binding site, or (iii) ATP may cause a conformational change that reduces binding of DNA.

Table 1.

K_d values for short DNA substrate saturation by MthMCM proteins (nM 12-mer)

	ssDNA		dsDNA
	−ATP	+ATP	−ATP	+ATP
WT	60 ± 21	65 ± 8	ND*	ND
R98A	>100	66 ± 30	428 ± 148	ND
Δ597	8 ± 2	>225	34 ± 8	ND
RAΔC	>100	>225	58 ± 16	ND

*ND K_d in all cases is >1 μM 12-mer.

Helicase Activity of MthMCM Mutants.

DNA unwinding was measured by strand displacement by using a 3′ tailed DNA substrate (Fig. 2). We observed that, in all cases, very high MCM to DNA ratios inhibited strand displacement. Surprisingly, despite the observed differences in ATPase activity and ssDNA affinity, the R98A and Δ597 proteins (Fig. 2 b and c) showed WT levels of helicase activity. Even more strikingly, despite having very low ATPase activity compared with WT, the combined RAΔC MthMCM mutant (Fig. 2d) was able to unwind a larger proportion of the helicase substrate at lower protein concentrations than either WT or the individual mutant proteins. To further address this point, we performed a time course experiment using a long substrate to examine the rate of duplex unwinding and the processivity of the different mutants. Fig. 3 shows that R98A is slightly more processive than WT over 30 min (compare lanes 20 and 8, respectively), whereas Δ597 is significantly more processive than WT. RAΔC displays the best processivity and has a faster rate of unwinding than Δ597 (compare 5-min time point, Fig. 3, lanes 10 and 23, respectively). In the context of our other results, this suggests that DNA unwinding by the MCM complex does not require high levels of ATP hydrolysis to be processive. Thus the N- and C-terminal motifs may act to modulate the efficiency with which ATP hydrolysis is utilized in DNA unwinding.

Fig. 2.

DNA unwinding activity is enhanced only in the RAΔC MthMCM mutant. Quantification of the amount of radiolabeled oligonucleotide displaced from a duplex possessing a 3′ tail by WT (a), R98A (b), Δ597 (c), and RAΔC (d) MthMCM proteins. Protein amounts are fmol of 12-mer complex in a 20-μl reaction.

Fig. 3.

Time course of helicase activity shows increased processivity of unwinding in Δ597 and RAΔC compared with WT. WT (lanes 5–8), R98A (lanes 17–20), Δ597 (lanes 22–25), or RAΔC (lanes 9–12) MCM complex (20 nM 12-mer) were incubated with 8 nM DNA substrate in the presence of 4 mM ATP for 1 min (lanes 5, 9, 17, and 22), 5 min (lanes 6, 10, 18, and 23), 10 min (lanes 7, 11, 19, and 24), or 30 min (lanes 8, 12, 20, and 25). Controls were labeled 24-residue oligonucleotide (lanes 1 and 14), sizing ladder (lanes 2 and 13), DNA substrate incubated on ice (lanes 3 and 15), boiled substrate (lanes 4 and 16), and mock-treated substrate (lane 21).

DNA- and Nucleotide-Induced Changes in Fluorescence.

The MthMCM protein contains only a single tryptophan residue (W363), located between the Walker A and B motifs of the central ATPase domain of the protein (Fig. 5a). To determine whether this region of the protein was influenced by the R98A and Δ597 mutations, we performed steady-state tryptophan fluorescence measurements. WT and mutant MCM proteins were incubated with nucleotide analogues or DNA, and the resulting signals were compared with those obtained in the absence of substrate (Fig. 4). Fluorescence changes consistent with the tryptophan being exposed to a more aqueous environment were observed in the WT protein on the addition of ATP, ADP, or nonhydrolyzable adenylyl-imidodiphosphate (AMP-PNP) (Fig. 4a). These changes were also observed in the R98A protein, although the change in signal between different analogues was less than observed for WT. The fluorescence changes were almost completely lost in the Δ597 protein (Fig. 4a). Combining both mutations produced an intermediate phenotype. The patterns of changes on addition of ssDNA or dsDNA (Fig. 4 b and c) were similar to those observed on nucleotide addition, although in all cases, addition of DNA resulted in larger fluorescence changes than the addition of nucleotides. In all cases, the Δ597 protein deviated most from WT and displayed changes consistent with the least movement in the locale of W363. These effects were largely overcome in the RAΔC double mutant. This suggests that the C-terminal domain of MCM exerts a strong conformational influence over the ATPase domain of the protein. Moreover, the environment around W363 is additionally modulated by the N-terminal domain of the protein, with the R98 residue also having a significant influence on the conformation of the central portion of the protein in a nucleotide- and DNA-dependent manner.

Fig. 4.

Movement of W363 in MthMCM. Relative changes in fluorescence measured on addition of nucleotide analogues (a), ssDNA (b), or dsDNA (c) to 0.3 μM protein are shown with an indication of whether the change is consistent with the environment of W363 becoming more or less hydrophobic. Bars are the average values from the measurement of at least three independent samples and are relative to the fluorescence observed in the absence of substrate. PNP, purine nucleoside phosphorylase.

Molecular Modeling of the Helix-2 Insert (h-2i).

We precisely located W363 in the MCM ATPase domain so we could investigate the specific role of the N- and C-terminal motifs on activity. Bioinformatic analysis of the AAA+ ATPase family suggests that MCMs can be differentiated from Tag and other superfamily 3 helicases by a h-2i between the Walker A and B motifs (26). This feature is not conserved at the level of amino acid sequence, but a β-α-β structural motif is predicted that would include W363 in MthMCM (Fig. 5a and b). The crystal structure of the AAA+ domain of magnesium chelatase, another h-2i clade protein, revealed that the h-2i forms a β-α-β loop positioned near the PS1BH (27). We generated a model of the N-terminal and ATPase domains of MCM from the magnesium chelatase ATPase structure and the N-terminal structure of MthMCM using the position of these domains proposed by Pape et al. (8) (Fig. 5 c and d). The model allowed us to visualize the putative location of the h-2i. It suggested that the h-2i was likely to interact with the PS1BH and protrude into the central channel of the MCM complex, potentially causing an obstruction to “loading” of substrate DNA. Thus our fluorescence measurements are likely reflecting the movement of the h-2i in response to nucleotide and DNA substrates.

Characterization of the MthMCM Δh-2i Mutant.

To determine whether the h-2i plays a role in MCM function, we deleted the 15 amino acids of the h2-i in MthMCM. The Δh2-i protein was soluble and formed high-molecular-weight complexes comparable to those previously described for MthMCM and consistent with a functional dodecamer (data not shown). ATPase activity of the mutant protein was compared with WT (Fig. 6a). In the presence of ssDNA and 50 μM ATP, the Δh-2i protein was comparable to WT, showing no stimulation of ATPase activity (1.09 ± 0.08 pmol ADP per min compared with 1.09 ± 0.18 pmol ADP per min for WT). However, in the presence of 50 μM ATP and dsDNA, Δh-2i showed a ≈12-fold increase in dsDNA-stimulated ATPase activity (41.48 ± 15.87 pmol ADP per min vs. 3.52 ± 0.57 pmol ADP per min for WT), confirming that the protein was functional and suggesting that the h-2i normally inhibits ATP hydrolysis.

Fig. 6.

Characterization of the MthMCM Δh-2i protein. (a) ATPase activity of WT or Δh2-i MthMCM in the absence of DNA or in the presence of 90 fmol closed circular ssDNA or 2.88 pmol dsDNA in 20-μl reactions. Values are hydrolysis rates for an input concentration of 50 μM ATP. (b) EMSA quantification (relative to input substrate) of ssDNA or (c) dsDNA-binding activity in the presence (open circles, dotted lines) or absence (closed circles, solid lines) of 4 mM ATP. Visualization of helicase activity for WT (d) or Δh2-i (e) MthMCM protein using strand displacement. Activity is indicated by the displacement of a labeled oligonucleotide from the substrate, resulting in a faster-migrating band. Boiled substrate controls indicate the position of the displaced oligonucleotide. The supershift observed in the samples incubated on ice is consistent with EMSA data. Protein amounts are fmol of a 12-mer complex in a 20-μl reaction.

DNA binding of Δh-2i was characterized by using EMSA. The mutant protein bound to both ssDNA and dsDNA substrates (Fig. 6 b and c) with much greater affinity than WT (Tables 1 and 2). In contrast to WT, where the presence of ATP caused a considerable increase in K_d, the presence of ATP actually reduced the K_d of Δh-2i for both DNA substrates. Using a strand displacement assay, we determined whether Δh-2i possessed DNA helicase activity. As reported (18–20), the WT protein displayed magnesium- and ATP-dependent DNA helicase activity in a concentration-dependent manner (Fig. 6d). In contrast, we were unable to detect any helicase activity for Δh-2i (Fig. 6e) despite its increased ATPase activity. Consistent with the EMSA results (Fig. 6 b and c), we observed a protein-dependent mobility shift of the helicase substrate when the reactions were incubated on ice. The proportion of DNA substrate shifted was greater in the presence of the Δh-2i mutant than in the presence of WT protein.

Table 2.

K_d values for short DNA substrate saturation by MthMCM Δh-2i protein (nM 12-mer)

	ssDNA		dsDNA
	−ATP	+ATP	−ATP	+ATP
Δh-2i	1.6 ± 0.2	0.7 ± 0.1	8.4 ± 3.4	3.7 ± 1.0

Our results strongly suggest that the MCM helix-2 β-α-β insert is essential for coupling the energy of ATP hydrolysis to DNA unwinding. In contrast to the Sulfolobus MCM PS1BH mutant (24), deletion of the h-2i from MthMCM caused a large increase in dsDNA-stimulated ATP hydrolysis and increased the affinity of the mutant complex for both ssDNA and dsDNA, even in the presence of ATP. The observed increase in ATP hydrolysis on deletion of the h-2i suggests that the MCM complex is capable of much higher levels of ATP hydrolysis than are normally observed for the WT protein in vitro.

Discussion

We have identified an N-terminal residue, R98, and a 72-aa C-terminal domain in MthMCM that affect both ATP hydrolysis and DNA binding. The mutations we have generated in these regions could reflect changes brought about in the MCM complex in vivo by protein–protein interactions or posttranslational modifications. Removal of both these influences resulted in an increased rate and processivity of DNA unwinding activity. Addition of substrates to the protein caused changes in the environment of the h-2i located between the Walker A and B motifs of the ATPase domain, consistent with conformational changes. The h-2i locale is additionally modulated by the N- and C-terminal motifs identified; these residues appear to have antagonistic effects on the h-2i in the presence of DNA or nucleotide. We predict that the h-2i protrudes into the central channel of the MCM complex alongside the PS1BH. The poor sequence conservation found in the h-2i relative to the surrounding AAA+ motifs suggests that whatever role this motif plays in MCM activity, it uses a mechanical mechanism that does not require specific amino acid interactions.

Removal of the h-2i from MthMCM resulted in the abrogation of DNA unwinding despite producing enhanced ATP hydrolysis. Thus the h-2i has a key role in the transduction of energy released by ATP hydrolysis into DNA unwinding activity. It should be emphasized that among helicases, the h-2i is unique to the MCM proteins: this motif is not present in other helicases such as Tag, and so Tag and the many other DNA helicases that fall outside the h-2i clade are likely to use a different mechanism for coupling ATP hydrolysis and unwinding. Our fluorescence results are consistent with movement of the h-2i into a more aqueous environment in the presence of substrates, suggesting this motif may move into the central channel of the MCM complex. In this case, the h-2i would be well positioned to mechanically separate the antiparallel strands of duplex DNA and act as the recently proposed molecular ploughshare (28). A scenario in which the PS1BH binds to the DNA substrate, followed by ATP-catalyzed opposing movement of the h-2i and the PS1BH in the central channel, is one mechanism by which the efficiency of duplex unwinding could be enhanced.

An enhanced ability to unwind DNA is potentially very important for the MCM helicase. The RAΔC mutant shows increased processivity of unwinding with decreased ATP hydrolysis, suggesting N-terminal components modulate the efficiency of transduction of ATP hydrolysis into duplex unwinding. Consistent with this hypothesis, increased processivity of the Archaeoglobus MCM was observed on deletion of 112 amino acids from the N terminus (29). Inhibition of the MthMCM complex by N-terminal interactions with CDC6 have also been recently described (30). The negative influence of both the C- and N-terminal domains on helicase activity may explain why eukaryotic complexes have not yet been shown to possess efficient helicase activity in vitro, a point that would benefit from further investigation in the light of these results.

Materials and Methods

Mutant Construction.

All mutants were generated by using the Stratagene Quik-Change site-directed mutagenesis (SDM) protocol. Oligonucleotide (Sigma Genosys) sequences are available on request. MthMCM amino acid positions were numbered from the second methionine in the sequence. pJC025 (18) was used as a template to produce R98A (pJC059), Δ597 (pLJ040), and Δh2-i (pLJ042). The double mutant, RAΔC (pLJ041), was produced by SDM of pJC059.

Protein Expression and Purification.

All constructs were expressed as described (18) and purified over TALON metal affinity resin (Clontech), followed by 50–500 mM NaCl elution from a Resource Q column (GE Biosciences). Purified proteins were desalted and spin-concentrated (30-kDa molecular weight cutoff, Vivaspin, Sartorius). Protein concentration was measured, and small aliquots were snap-frozen before being stored at −80°C.

ATP Hydrolysis Assay.

Assays were performed as described (18). ATP was mixed with [α-³²P]-ATP [3,000 Ci/mmol (1 Ci = 37 GBq), GE Biosciences] and titrated into 20-μl reactions containing 4.32 pmol monomeric protein. Reactions contained no DNA or were supplemented with either 2.88 pmol closed circular dsDNA or 90 fmol closed circular ssDNA. The data were calibrated by using a dilution series of the ATP stock performed in parallel with the experiment. Signals were detected by phosphorimaging and quantified by using Quantity One (Bio-Rad).

EMSA.

Experiments were performed as described (18) by using 2.5 nM 5′ radiolabeled 30-residue oligonucleotide as the ssDNA substrate. The dsDNA substrate was made by annealing the labeled oligonucleotide to a second oligonucleotide with complementary sequence and added to 2.5 nM. ATP was added to a final concentration of 4 mM as appropriate. Dissociation constants (K_d) were determined where saturation was reached by fitting a single rectangular 2 parameter hyperbola (Eq. 1) to the data using sigmaplot 9 (Systat, Point Richmond, CA). In all cases, R² ≥ 0.99.

Helicase Assays.

Strand-displacement assays were performed as described (18) with the following modifications: the substrate was made by labeling a 26-residue oligonucleotide with [γ-³²P]-ATP (GE Biosciences) using T4 polynucleotide kinase (New England Biolabs). The labeled oligonucleotide was desalted and annealed to a 50-residue oligonucleotide with sequence complementary to the labeled oligonucleotide. This resulted in a substrate of 26 base pairs and a 24-nt 3′ tail. Twenty-microliter reactions containing 4 mM ATP and 20 fmol substrate were incubated at 50°C for 30 min before being stopped and separated on a 15% polyacrylamide (19:1) gel in 1× TBE (90 mM Tris/90 mM boric acid/2 mM EDTA, pH 8.0). Processivity assays were performed by using a substrate based on the work of Tabor and Richardson (18, 31) and are described in detail elsewhere (S. Castella, D. Burgin, and C. M. Sanders, personal communication).

Fluorescence Measurements.

Control and sample reactions containing 0.5 mM nucleotide or different concentrations of 30-residue oligonucleotide were incubated with 0.3 μM protein for 5 min at room temperature before being excited at 295 nm in a FluoroMax-2 fluorimeter (Jobin Yvon, Longjumeau, France). Emission spectra (310–400 nm) were collected and the data averaged from at least four technical and three biological replicates. Values at 340 nm were compared. Scans of blank samples showed no inner filter effects on addition of nucleotide or DNA alone.

Molecular Modeling.

The Protein Data Bank (PDB) file for magnesium chelatase (PDB ID code 1G8P) was edited to remove regions that were not homologous to MthMCM before being fitted to the electron density map of MthMCM (8). The N terminus of MthMCM (1LTL) was docked in a similar way, and the resulting model was visualized by using macpymol, Ver. 0.99 (DeLano Scientific, South San Francisco, CA). The h-2i was identified by using clustalx (32) alignments of the amino acid sequences of the appropriate regions of magnesium chelatase and MthMCM (26).

Abbreviations

MCM

minichromosome maintenance

MthMCM

Methanothermobacter thermautotrophicus MCM

h-2i

helix-2 insert

PS1BH

presensor 1 β-hairpin in the ATPase domain.