Physical Interactions between Mcm10, DNA, and DNA Polymerase α*

Mcm10 is an essential eukaryotic protein required for the initiation and elongation phases of chromosomal replication. Specifically, Mcm10 is required for the association of several replication proteins, including DNA polymerase α (pol α), with chromatin. We showed previously that the internal (ID) and C-terminal (CTD) domains of Mcm10 physically interact with both single-stranded (ss) DNA and the catalytic p180 subunit of pol α. However, the mechanism by which Mcm10 interacts with pol α on and off DNA is unclear. As a first step toward understanding the structural details for these critical intermolecular interactions, x-ray crystallography and NMR spectroscopy were used to map the binary interfaces between Mcm10-ID, ssDNA, and p180. The crystal structure of an Mcm10-ID·ssDNA complex confirmed and extended our previous evidence that ssDNA binds within the oligonucleotide/oligosaccharide binding-fold cleft of Mcm10-ID. We show using NMR chemical shift perturbation and fluorescence spectroscopy that p180 also binds to the OB-fold and that ssDNA and p180 compete for binding to this motif. In addition, we map a minimal Mcm10 binding site on p180 to a small region within the p180 N-terminal domain (residues 286–310). These findings, together with data for DNA and p180 binding to an Mcm10 construct that contains both the ID and CTD, provide the first mechanistic insight into how Mcm10 might use a handoff mechanism to load and stabilize pol α within the replication fork.

To maintain their genomic integrity, cells must ensure complete and accurate DNA replication once per cell cycle. Consequently, DNA replication is a highly regulated and orchestrated series of molecular events. Multiprotein complexes assembled at origins of replication lead to assembly of additional proteins that unwind chromosomal DNA and synthesize nascent strands. The first event is the formation of a pre-replicative complex, which is composed of the origin recognition complex, Cdc6, Cdt1, and Mcm2-7 (for review, see Ref. 1). Initiation of replication at the onset of S-phase involves the activity of cyclin-and Dbf4-dependent kinases concurrent with recruitment of key factors to the origin. Among these, Mcm10 (2, 3) is recruited in early S-phase and is required for loading of Cdc45 (4). Mcm2-7, Cdc45, and the GINS complex form the replicative helicase (5)(6)(7)(8). Origin unwinding is followed by loading of RPA, 3 And-1/Ctf4, and pol ␣ onto ssDNA (9 -12). In addition, recruitment of Sld2, Sld3, and Dpb11/TopBP1 are essential for replication initiation (13,14), and association of topoisomerase I, proliferating cellular nuclear antigen (PCNA), replication factor C, and the replicative DNA polymerases ␦ and ⑀ completes the replisome (for review, see Ref. 15).
Mcm10 is exclusive to eukaryotes and is essential to both initiation and elongation phases of chromosomal DNA replication (6,8,16). Mutations in Mcm10 in yeast result in stalled replication, cell cycle arrest, and cell death (2,3,(17)(18)(19). These defects can be explained by the number of genetic and physical interactions between Mcm10 and many essential replication proteins, including origin recognition complex, Mcm2-7, and PCNA (3, 12, 20 -24). In addition, Mcm10 has been shown to stimulate the phosphorylation of Mcm2-7 by Dbf4-dependent kinase in vitro (25). Thus, Mcm10 is an integral component of the replication machinery.
Importantly, Mcm10 physically interacts with and stabilizes pol ␣ and helps to maintain its association with chromatin (16,26,27). This is a critical interaction during replication because pol ␣ is the only enzyme in eukaryotic cells that is capable of initiating DNA synthesis de novo. Indeed, Mcm10 stimulates the polymerase activity of pol ␣ in vitro (28), and interestingly, the fission yeast Mcm10, but not Xenopus Mcm10, has been shown to exhibit primase activity (29,30). Mcm10 is composed of three domains, the N-terminal (NTD), internal (ID), and C-terminal (CTD) domains (29). The NTD is presumably an oligomerization domain, whereas the ID and CTD both interact with DNA and pol ␣ (29). The CTD is not found in yeast, whereas the ID is highly conserved among all eukaryotes. The crystal structure of Mcm10-ID showed that this domain is com-posed of an oligonucleotide/oligosaccharide binding (OB)-fold and a zinc finger motif, which form a unified DNA binding platform (31). An Hsp10-like motif important for the interaction with pol ␣ has been identified in the sequence of Saccharomyces cerevisiae Mcm10-ID (16,26).
DNA pol ␣-primase is composed of four subunits: p180, p68, p58, and p48. The p180 subunit possesses the catalytic DNA polymerase activity, and disruption of this gene is lethal (32,33). p58 and p48 form the DNA-dependent RNA polymerase (primase) activity (34,35), whereas the p68 subunit has no known catalytic activity but serves a regulatory role (36,37). Pol ␣ plays an essential role in lagging strand synthesis by first creating short (7-12 nucleotide) RNA primers followed by DNA extension. At the critical length of ϳ30 nucleotides, replication factor C binds to the nascent strand to displace pol ␣ and loads PCNA with pols ␦ and ⑀ (for review, see Ref. 38).
The interaction between Mcm10 and pol ␣ has led to the suggestion that Mcm10 may help recruit the polymerase to the emerging replisome. However, the molecular details of this interaction and the mechanism by which Mcm10 may recruit and stabilize the pol ␣ complex on DNA has not been investigated. Presented here is the high resolution structure of the conserved Mcm10-ID bound to ssDNA together with NMR chemical shift perturbation competition data for pol ␣ binding in the presence of ssDNA. Collectively, these data demonstrate a shared binding site for DNA and pol ␣ in the OB-fold cleft of Mcm10-ID, with a preference for ssDNA over pol ␣. In addition, we have mapped the Mcm10-ID binding site on pol ␣ to a 24-residue segment of the N-terminal domain of p180. Based on these results, we propose Mcm10 helps to recruit pol ␣ to origins of replication by a molecular hand-off mechanism.

EXPERIMENTAL PROCEDURES
Protein Expression and Purification-Mcm10-ID was prepared as described previously (31). Briefly, the protein was overexpressed from a modified pET-32a vector (Novagen) in Escherichia coli BL21(DE3) cells for 16 h at 16°C and isolated using nickel affinity chromatography. After cleavage of the thioredoxin-His 6 tag, Mcm10-ID was purified using ssDNA affinity and size exclusion chromatography. An Mcm10 construct spanning amino acid residues 230 -860 (Mcm10-IDϩCTD) was cloned and expressed similarly to Mcm10-ID, except protein expression was induced at 21°C for 4 h. Mcm10-IDϩCTD protein was purified by nickel-nitrilotriacetic acid affinity chromatography (Qiagen) followed by S-Sepharose (GE Healthcare) ion exchange chromatography and cleavage of the affinity tag. The cleaved protein was further purified by gel filtration using a Superdex 200 preparative column (GE Healthcare) equilibrated in 20 mM Tris, pH 7.5, 150 mM NaCl, 3.5 mM ␤-mercaptoethanol, and 5% glycerol (buffer A).
The DNA encoding amino acids 189 -323 of human p180 was ligated into a modified pET-27 vector (Novagen) to produce an N-terminal His 6 fusion protein (pBG100, Vanderbilt Center for Structural Biology). E. coli BL21(DE3) cells transformed with the p180 189 -323 /pBG100 plasmid were grown at 37°C in LB medium containing 100 g/ml ampicillin, and protein was overexpressed by the addition of 0.5 mM isopropyl 1-thio-␤-D-galactopyranoside for 4 h. For NMR experiments, protein was uniformly enriched with 15 N by propagating cells in M9 minimal medium supplemented with 1 mg/ml 15 NH 4 Cl (Cambridge Isotope Laboratories) as the sole source of nitrogen. The cells were harvested in 50 mM Tris, pH 7.5, 500 mM NaCl, and 10% glycerol and lysed under pressure (25,000 p.s.i.) using an Avestin EmulsiFlex C3 homogenizer. p180 189 -323 was purified by nickel-nitrilotriacetic acid affinity chromatography (Qiagen) followed by cleavage of the affinity tag. The cleaved protein was further purified by Q-Sepharose (GE Healthcare) ion exchange chromatography followed by gel filtration using a Superdex 200 preparative column (GE Healthcare) equilibrated in buffer A. p180 243-256 and p180 286 -310 peptides used for NMR titrations were synthesized and purified by Genescript Corp. (Piscataway, NJ).
X-ray Crystallography-Crystals were grown by sitting drop vapor diffusion by mixing 2 l of protein/DNA solution containing 300 M Mcm10-ID and 360 M dC 9 ssDNA with 2 l of reservoir solution containing 100 mM TAPS, pH 9.0, and 17% polyethylene glycol 3350. Crystals appeared overnight and grew to ϳ50 ϫ 50 ϫ 200 m 3 after 2-3 days. Crystals were soaked for 5 min in mother liquor containing 10% (v/v) butanediol and flash-frozen in liquid nitrogen. Preliminary x-ray diffraction data (Table 1) were collected at beamline 21-ID at the Advanced Photon Source (Argonne, IL) and processed with HKL2000 (39). The Mcm10-ID⅐ssDNA complex crystallized in space group P3 1 21 with one molecule in the asymmetric unit.
X-ray phases were obtained by molecular replacement using unliganded Mcm10-ID (PDB code 3EBE) as the search model in the program Molrep (40). A clear rotation/translation solution was verified by the quality of the resulting composite annealed 2F o Ϫ F c omit electron density maps generated using CNS (41) (supplemental Fig. S1). Several iterative rounds of restrained atomic and temperature factor refinement against a maximum likelihood crystallographic target in Phenix (42) together with manual model adjustment and building the L12 loop in Coot (43) resulted in R and R free values of 21.7 and 26.1%, respectively. Strong F o Ϫ F c difference Fourier density was observed within the OB-fold cleft (residues 292-360). Three nucleotides of ssDNA were fit into this density using Coot and refined, which lowered R and R free by 0.8 and 1.61%, respectively. The polarity of the DNA was established by parallel refinement of both orientations of DNA, which differed from each other by 1% in R free . Translation/libration/screw refinement was used to model anisotropic motion of 6 groups, defined by protein residues 235-241, 242-260, 261-300, 301-371, 372-500, and DNA as determined by the TLSMD server (44). Individual anisotropic B-factors were derived from the refined translation/libration/screw parameters and held fixed during subsequent rounds of refinement, which resulted in a noticeable improvement of the electron density maps (supplemental Fig.  S1) and a 1% decrease in both R and R free . No additional electron density was discerned corresponding to the six remaining nucleotides or for residues 230 -231 and 416 -427 at the extreme N and C termini.
Analysis of the final structure using PROCHECK (45) showed 80.5 and 19.5% of the total of 154 non-glycine and nonproline residues to be within the most favored and allowed regions of the Ramachandran plot, respectively, with no resi-dues in the disallowed region. The coordinates and structure factors have been deposited in the Protein Data Bank under accession number 3H15.
NMR Spectroscopy-Gradient-enhanced 1 H, 15 N HSQC NMR spectra were recorded at 25°C using a Bruker DRX 800 NMR spectrometer equipped with single axis z-gradient cryoprobe. All spectra were acquired with 1024 complex points over a sweep width of 15 ppm in the 1 H dimension and 128 complex points over 37 ppm in the 15 N dimension. The center of the 15 N spectral width was set to 117.5 ppm, and the 1 H carrier was placed on the water signal at 4.7 ppm from the respective base spectrometer frequencies. All spectra were processed and analyzed using Topspin v1.3 (Bruker, Billerica, MA) and Sparky version 3.1 (University of California, San Francisco, CA). Data were treated with shifted sine-bell functions and zero-filled to twice the number of data points in both dimensions.
Chemical shift perturbation data were collected by titrating 1. Fluorescence Anisotropy-Mcm10-ID and Mcm10-IDϩCTD binding to p180 and DNA was measured by following an increase in fluorescence anisotropy as unlabeled Mcm10 domains were added to fluorescein labeled p180 fragments and oligonucleotides. Fluorescein-p180 189 -323 was prepared by incubating purified p180 189 -323 with a 20-fold molar excess of MTS-fluorescein (2-[(5-fluoreceinyl) aminocarbonyl]ethyl methanethiosulfonate, Toronto Research Chemicals) at 25°C for 6 h followed by purification on a 1-ml Q-Sepharose column (GE Healthcare). Fluorescein isothiocyanate-conjugated p180 286 -310 was synthesized and purified by Genescript Corp., and oligonucleotides containing 3Ј-labeled 6-carboxyfluorescein were synthesized by Integrated DNA Technologies. All binding and competition reactions were carried out at 25°C in 20 mM Tris, pH 7.5, 150 mM NaCl, and 5% glycerol. Polarized fluorescence intensities were measured at excitation and emission wavelengths of 495 and 538 nm, respectively. For binding measurements, unlabeled Mcm10 protein was added over a concentration range of 0.1-50 M to solutions containing either 50 nM fluorescein-p180 fragment or 50 nM fluorescein-DNA. Dissociation constants (K d ) were derived by fitting a twostate binding model to data from three experiments using Kaleidagraph 3.6 (Synergy Software) according to the equation  Table 2). Under these conditions, both fluorescein-labeled molecules are Ͼ80% saturated ( Isothermal Titration Calorimetry-Proteins were buffer-exchanged into 25 mM Tris, pH 7.5, and 100 mM NaCl and concentrated to 50 M (Mcm10-ID) and 1 mM (p180 189 -323 ). 1.7 ml of Mcm10-ID was placed in the sample cell, into which p180 189 -323 was injected in 6-l steps during the run. Data were collected at 25°C using a MicroCal VP-isothermal titration calorimetry and analyzed using the accompanying Origin software (Origin Lab, Northampton, MA). Thermodynamic parameters were calculated from fitting the data to the best binding model using Origin according to the Gibbs free energy equation, ⌬G ϭ ⌬H Ϫ T⌬S ϭ ϪRTln K a .

RESULTS
The Crystal Structure of Mcm10-ID Bound to ssDNA-The highly conserved internal domain of Xenopus laevis Mcm10 (Mcm10-ID) has previously been suggested to bind DNA along the surface formed by the concave OB-fold ␤-barrel and the extended zinc finger loop (31). To elucidate the details of the Mcm10-ID⅐ssDNA interaction at high resolution, we determined the crystal structure of Mcm10-ID in complex with ssDNA using the unliganded Mcm10-ID structure as a molecular replacement search model (Fig. 1). Strong electron density corresponding to three consecutive cytidine nucleotides of ssDNA was clearly visible inside the OB-fold cleft (Fig. 1A), similar to the location of bound ssDNA in OB-fold structures of RPA, Rho, RecG, and RumA (46 -50). These proteins typically bind ssDNA between the OB-fold L12 and L45 loops, which are often flexible in the absence of DNA (51). Consistent with other OB-fold⅐ssDNA complexes, Mcm10-ID L12 loop (residues 297-305), which was not observed in the unliganded structure, is now visible in the complex as the flexibility of this loop is quenched by interactions with the DNA (Fig. 1B). The atomic model for Mcm10-ID⅐ssDNA was refined to 2.7 Å to a crystallographic residual of 19.7% (R free ϭ 23.2%). Data collection and refinement statistics are shown in Table 1.
The electron density for the ssDNA traverses ␤-strands ␤1-␤3 and ␤5.1, which form the concave surface of the OB-fold cleft (Fig. 1A). The polarity of the DNA is such that the 5Ј-end starts at the ␤5.1 strand and the 3Ј-end points toward ␤1 and the zinc finger in a similar manner to the RPA70AB⅐ssDNA complex (46). Refining the DNA in the opposite orientation had a detrimental impact on the crystallographic residual. The L12 and L45 loops wrap around the DNA, creating a channel ϳ16 Å in diameter (Fig. 1B). Polar and hydrophobic side chains from both loops and lining the ␤-sheet make van der Waals contact with the DNA, including Ser-299, Phe-306, Ile-308, Phe-324, Phe-326, Met-350, and Lys-353 ( Fig. 1C and supplemental Fig.  S2). The high B-factors for the DNA (Table 1) indicate that the DNA is highly mobile and somewhat disordered within this hydrophobic cleft, which precludes our ability to precisely model the DNA atoms that contact the protein. Nonetheless, the DNA binding surface on the protein and the polarity of the DNA are clearly defined. Electron density was not observed for the DNA around the zinc finger, presumably because of steric occlusion of the zinc finger binding site due to crystal packing. In this crystal form, the L45 loop of a neighboring molecule protrudes into the cleft between the OB-fold and the zinc finger, blocking ssDNA access to the zinc finger (supplemental Fig. S3). Although the entire DNA molecule cannot be identified from the present crystallographic data, this result confirms previous evidence that ssDNA binds directly to the OB-fold cleft (31) and is consistent with the orientation of DNA observed in other OB-folds (46 -48).
The structure of ssDNA-bound Mcm10-ID is nearly identical to the unliganded structure previously published with root mean square deviation of 0.77Å for all C␣ atoms (31). Apart from the now-ordered L12 DNA binding loop, the only notable difference between the two structures lies at the zinc finger helix at the extreme C terminus of the ID (residues 405-416). This helix is well defined in the unliganded protein and is engaged in intermolecular protein-protein contacts in each of the three protomers in the asymmetric unit (supplemental Fig. S4) (31). In the complex, which crystallizes in a different lattice with one protein-DNA complex per asymmetric unit, the zinc finger helix is disordered past the Zn 2ϩ -coordinating His-406. This local unfolding is presumably because of the lack of any intermolecular contacts in the present crystal lattice and suggests that the fold of this helix in the full-length protein may be stabilized through protein contacts outside of the ID.
Mcm10-ID Binds to p180 189 -323 -Mcm10-ID has previously been shown to bind to the N-terminal 323 residues of the p180 subunit of human pol ␣-primase (29). This region is highly conserved but lacks appreciable predicted secondary structure or sequence complexity. To map the Mcm10-p180 interaction in detail, p180 1-323 was subjected to limited proteolysis, and the resulting stable fragments were identified by mass spectrometry. Proteolytically sensitive sites were found at residues 145 and 189. Consequently, p180 1-145 and p180 189 -323 were subcloned, purified, and tested for physical interaction with Mcm10-ID by affinity chromatography pulldown assays. The p180 1-145 protein was not sufficiently stable in solution to test for a putative interaction. However, GST-tagged p180 189 -323 immobilized on glutathione-Sepharose was able to capture Histagged Mcm10-ID from solution ( Fig. 2A), demonstrating that this region of the p180 subunit is sufficient to bind to Mcm10-  ID. The strength of the Mcm10-ID-p180 189 -323 interaction was quantified using a fluorescence anisotropy assay. Titration of unlabeled Mcm10-ID into a solution of fluorescein-labeled p180 189 -323 resulted in a robust increase in fluorescence anisotropy, whereas the addition of either ssDNA or buffer alone had no effect (Fig. 2B). Analysis of the titration data by a twostate binding model provided an apparent dissociation constant (K d ) of 12 Ϯ 2 M for Mcm10-ID binding to p180 189 -323 . This value is in good agreement with the K d (30 Ϯ 1 M) determined by isothermal titration calorimetry using unlabeled proteins (Fig. 2C). NMR chemical shift perturbation experiments were used to probe the Mcm10-ID and p180 189 -323 interaction in greater detail and map the p180 189 -323 binding site on Mcm10-ID. We previously obtained sequence specific backbone assignments of Mcm10-ID and used chemical shift perturbation to map the DNA binding site (31). Here, we monitored 1 H and 15 N chemical shift perturbations of uniformly 15 N-labeled Mcm10-ID upon the addition of unlabeled p180 189 -323 (Fig. 3A). These experiments revealed a number of significant chemical shift perturbations in the two-dimensional 1 H, 15 N HSQC spectrum that mapped onto the OB-fold cleft, with the strongest pertur-bations observed for residues in ␤1, ␤2, ␤5.1, L12, and L45 (Fig. 3, B and  C). In fact, the resulting spectrum for the Mcm10-ID⅐p180 189 -323 complex is remarkably similar to that previously measured for Mcm10-ID⅐ssDNA complex (31) (supplemental Fig. S9), suggesting that Mcm10-ID utilizes a common binding site for both ssDNA and p180. Moreover, both p180 189 -323 and ssDNA bind to Mcm10-ID in the fast-to-intermediate-exchange regime, with some peaks gradually shifting over the course of the titration, whereas others broaden and disappear. However, by comparing the magnitude of the chemical shift perturbations in response to p180 189 -323 and ssDNA binding, it appears that Mcm10-ID binds more weakly to p180 189 -323 than to ssDNA (data not shown). This observation is consistent with the 4-fold weaker Mcm10 dissociation constant determined by fluorescence anisotropy (3 Ϯ 1 M for ssDNA versus 12 Ϯ 2 M for p180 189 -323 ).
ssDNA and p180 189 -323 Compete for the Same Site on Mcm10-ID-A common binding site for ssDNA and p180 suggests that these two ligands either compete for binding or bind cooperatively to Mcm10. To distinguish between these two possibilities, competition experiments were performed utilizing NMR chemical shift perturbations to monitor the interaction of p180 189 -323 and ssDNA with Mcm10-ID (Fig. 4A, supplemental Fig. S5A). First, ssDNA was titrated into a sample containing 15 N-labeled Mcm10-ID, and peak shifts were observed as previously reported (Fig. 4A, red spectrum) (31). Next, unlabeled p180 189 -323 was titrated into the same sample containing ssDNA-saturated Mcm10-ID (Fig. 4A, green spectrum). No further chemical shift perturbations were observed with the addition of protein, which suggests that p180 189 -323 neither interacts with an Mcm10-ID⅐ssDNA complex nor does it displace ssDNA from Mcm10-ID.
To test whether ssDNA is able to disrupt a preformed Mcm10-ID⅐p180 189 -323 complex, the reverse titration was performed in which unlabeled p180 189 -323 was first added to a sample containing 15 N-labeled Mcm10-ID followed by the addition of ssDNA (Fig. 4B, supplemental Fig. S5B). As in Fig. 3, the addition of p180 189 -323 to 15 N-labeled Mcm10-ID resulted in significant perturbation in chemical shifts for a discrete set of residues (Fig. 4B, blue spectrum). Upon the addition of ssDNA, the peaks that were perturbed by p180 189 -323 changed trajectory and shifted to resemble the Mcm10-ID⅐ssDNA spectrum (Fig. 4B, gold spectrum). To test the ability of ssDNA to displace Mcm10 from p180, we performed a NMR titration in which Mcm10-ID and ssDNA were added in succession to a solution containing 15 N-enriched p180 189 -323 . When Mcm10-ID was titrated into 15 N-p180 189 -323 , displacement of a discrete number of chemical shifts was observed, indicative of formation of the Mcm10-ID⅐p180 complex (Fig. 4C, supplemental Fig. S6A, blue spectrum). The addition of ssDNA to the protein complex caused the chemical shifts to revert to their starting location in the spectrum of 15 N-p180 189 -323 alone (Fig. 4C,  gold). This directly demonstrates that ssDNA is capable of displacing Mcm10-ID from p180 189 -323 . A fourth titration was performed in which ssDNA was first added into the 15 Nlabeled p180 189 -323 sample. In this case no peak shifts were observed, indicating that p180 189 -323 does not bind ssDNA (Fig. 4D, supplemental Fig. S6B, green spectrum). When Mcm10-ID was titrated into this sample containing free p180 189 -323 and ssDNA, perturbation of p180 chemical shifts that mimicked the 15 N-p180 189 -323 /Mcm10-ID spectrum were observed (compare the blue spectrum in Fig. 4C with the red spectrum in Fig. 4D). However, the magnitude of Mcm10-ID-induced 15 N-p180 189 -323 peak shifts in the presence of ssDNA were not as large as those in the absence of ssDNA, consistent with a partitioning of Mcm10 between both p180 189 -323 and ssDNA. Taken together, these data demonstrate that ssDNA and p180 189 -323 compete for binding to the OB-fold cleft of Mcm10-ID and that ssDNA is able to displace p180 189 -323 from Mcm10-ID, consistent with the moderate preference of Mcm10-ID for ssDNA over p180 189 -323 .
To quantify the competition of ssDNA and p180 189 -323 for Mcm10-ID, we examined the concentration dependence on the displacement reaction using the fluorescence anisotropy assay  Fig. S6) was carefully examined to determine whether insights could be obtained into the location of the Mcm10-ID binding site on p180 189 -323 , even in the absence of sequence specific assignments, following the strategy described previously (52). The key to this approach is to monitor the total number of resonances perturbed in the titration and take advantage of the unique chemical shifts of the glycine backbone and glutamine and asparagine side chain amides. Analysis of the data in this way suggests that the binding sequence should contain ϳ20 residues including at least one glycine and no asparagine or glutamine residues. In the sequence of p180 189 -323 , only two peptides fit these criteria, p180 243-256 and p180 286 -310 . Indeed, glutathione-immobilized GST-p180 243-310 , which spanned both peptides, was able to capture Mcm10-ID from solution in our affinity chromatography assay ( Fig. 2A). To  6A, supplemental Fig. S9). Binding of p180 286 -310 occurred on the fast-exchange timescale and resulted in a magnitude of chemical shift perturbations similar to those caused by p180 189 -323 (supplemental Fig. S7). These data show that p180 residues 286 -310 bind to the same region of Mcm10-ID as p180 189 -323 and ssDNA (Fig. 6, B and C) and are consistent with the relative binding affinities for p180 189 -323 (K d ϭ 12 Ϯ 2 M) and p180 286 -310 (K d ϭ 32 Ϯ 2 M) measured by fluorescence anisotropy (supplemental Fig. S7D).
Mcm10-IDϩCTD Binds ssDNA and p180 189 -323 -Having thoroughly characterized the binding of ssDNA and p180 to Mcm10-ID, we asked how studies of the isolated domain relate to the biochemical functions of the intact protein. Mcm10-CTD has previously been shown to bind both DNA and p180   (29). The linking of the ID and CTD are, therefore, anticipated to result in higher affinity and possibly altered specificity. To this end, a protein deletion construct encompassing Mcm10-ID and -CTD was constructed, purified, and characterized by biochemical approaches.
Binding of Mcm10-IDϩCTD to various DNA substrates designed to resemble replication intermediates was measured by fluorescence anisotropy ( Table 2). The goal of these experiments was to test the effects of DNA structure and length under identical conditions and to compare the relative affinities between DNA and p180 (see below). Mcm10-IDϩCTD bound all DNAs tested with 10-fold greater affinity than previously determined for ID or CTD alone. This observation is similar to our previous results obtained with a maltose-binding proteintagged full-length Mcm10 (29). Additionally, the IDϩCTD protein bound ssDNA with a slightly higher affinity than dsDNA when tested against both 25-and 45-mer oligonucleotides, as observed previously for the isolated domains and the intact protein (28,29,31) (Table 2). Moreover, Mcm10-IDϩCTD does not demonstrate a significant preference for ssDNA, dsDNA, or constructs containing ss/dsDNA junctions, including 5Ј-and 3Ј-overhangs, fork, and bubble substrates ( Table 2). This lack of specificity for a particular DNA structure was observed previously for the isolated ID and CTD. Thus, together these two binding modules enhance the strength of the DNA interaction but do not provide additional specificity.
Binding  Table 2). The strength of the Mcm10-IDϩCTD interaction with p180 189 -323 is ϳ50fold greater than that measured for Mcm10-ID alone (K d ϭ 12 Ϯ 2 M). Importantly, the affinity observed for this tandem construct brings the strength of binding of p180 to the same level as for ssDNA (Table 2). This has important implications for how Mcm10 might recruit p180 (and therefore pol ␣) to the active replication machinery.

DISCUSSION
Chemical Nature of Mcm10-ID Interactions with DNA and Pol ␣-In this study we show that both ssDNA and the N-terminal region of p180 compete for binding to a relatively hydrophobic surface within the OB-fold cleft of Mcm10. Our previous analysis showed that in addition to the OB-fold, ssDNA binds to the highly basic extended loop on the zinc finger motif (31). In the structure of the Mcm10⅐ssDNA complex, the crystal lattice prevented DNA access to the zinc loop, which precluded direct visualization of the interaction between DNA and the zinc finger. However, additional information regarding the nature of intermolecular Mcm10-ID interactions can be inferred from thermodynamic information derived from isothermal titration calorimetry measurements. Titration of Mcm10-ID with ssDNA (supplemental Fig. S2B) revealed an enthalpy-driven, spontaneous reaction (⌬H ϭ Ϫ9.8 kcal/mol, T⌬S ϭ Ϫ3.6 kcal/mol). This is consistent with our previous mutational analysis that showed electrostatic interactions play a large role in ssDNA binding to Mcm10-ID (31). Taking the structural and biochemical data together, binding of ssDNA to Mcm10-ID is largely mediated by hydrophobic residues located within the OB-fold cleft as well as by polar/charged residues located on the L12 and L45 loops (e.g. Ser-299) between the OB-fold and the zinc finger (Lys-293) and on the zinc loop (Lys-385 and Lys-386). In contrast, calorimetric titration of Mcm10-ID with p180 189 -323 (Fig. 2C) revealed a large entropic contribution (⌬H ϭ 0.4 kcal/mol; T⌬S ϭ 6.5 kcal/mol), suggesting that hydrophobic interactions may be important to the proteinprotein interaction. Indeed, p180 189 -323 and p180 286 -310 binding mapped to the aliphatic OB-fold cleft. Interestingly, NMR chemical shifts corresponding to basic residues on the zinc finger helix (␣E) and not the DNA binding zinc loop were perturbed by all three p180 constructs tested, including p180 243-256 , which did not bind to the OB-fold (supplemental Fig. S9). Thus, we speculate that a hydrophobic interaction at the OB-fold may provide additional specificity for p180 286 -310 .
Pol ␣ and Mcm10 Binding Domains-The overall domain structure of p180 is known, and the activities of the central polymerase and the C-terminal subunit assembly domains have been characterized (53,54). However, the function of the N-terminal domain is less clear. This region of p180 is dispensable for polymerase activity and is not required for assembly of the pol ␣-primase complex. The N terminus of p180 is phosphorylated by cyclin A-dependent kinase 2 on residues 174, 209, and 219 (55,56), and it interacts with several proteins of various functions including Mcl1 (And-1/Ctf4) (57), PP2A (56), concanavalin A and Ricinus communis agglutinin I (58), SV40 Large T-antigen (59,60), and Mcm10 (29). Although the importance of these interactions has yet to be determined, our observation that Mcm10 interacts with p180 286 -310 outside of the polymerase domain is consistent with Mcm10 anchoring the pol ␣ complex onto DNA in such a way as to not interfere with RNA or DNA synthesis.
The finding that Mcm10-ID interacts with both ssDNA and pol ␣ through contacts in the OB-fold domain reflects the adaptability of this motif to bind a range of different biological molecules. This cleft is used by various proteins to engage RNA (61), DNA (46), oligosaccharides (62), proteins (63), and even metals and inorganic phosphates (64,65). For example, RPA, a eukaryotic recruiting and scaffolding protein critical to DNA replication, has been shown to bind both oligonucleotides and peptides through its six OB-fold domains (66 -70). Mcm10 appears to exhibit similar behavior by binding to DNA, pol ␣, Dbf4-dependent kinase, and PCNA (24,25,31), although the role of the OB-fold in Mcm10 interactions with Dbf4-dependent kinase and PCNA remains to be determined.
A Molecular Mechanism for Mcm10 Hand-off of Pol ␣ to DNA-This is the first report of competition between DNA and pol ␣ for binding to Mcm10. Competition for sites provides a ready mechanism for direct coupling of the protein interaction with DNA binding as a means to promote progression of the replication machinery. Although the exact role of Mcm10 in replication initiation has yet to be elucidated, it is reasonable to envision Mcm10 as a macromolecular recruiting and/or scaffolding protein because of the fact that Mcm10 contains two domains that can bind to DNA and pol ␣ (29). This follows a common strategy for numerous modular proteins involved in DNA processing; there is a significant kinetic advantage to deconstructing protein interactions into two or more weak binding sites (68). The recruitment of pol ␣ to origins of replication by Mcm10 would be a significant step to signal nascent DNA synthesis and contribute to fork stability (6,12,16,24,26,27,29,71). Indeed, Mcm10 has been shown to be necessary for pol ␣ loading onto chromatin (4).
The detailed analysis of binding affinities and competition experiments presented here demonstrate that the highly conserved Mcm10-ID transitions between interaction with DNA and pol ␣, consistent with an Mcm10-mediated hand-off mechanism (Fig. 7). The relatively similar affinities of p180 189 -323 and ssDNA for Mcm10-IDϩCTD suggest that full-length Mcm10 also partitions between DNA and pol ␣ binding. Two scenarios for hand-off of pol ␣ onto DNA by a single Mcm10 molecule can be envisioned, the first in which the CTD interacts with ssDNA as the ID engages p180 286 -310 (Fig. 7A). Equiv-alently, Mcm10 could bind to the DNA through the ID, while the CTD tethers the N-terminal region of p180 (Fig. 7B). It is interesting to note that binding of p180 189 -323 to CTD alone was undetectable by our fluorescence assay (data not shown), raising the possibilities that either the CTD binds to p180 1-323 outside of the 189 -323 subdomain or that the CTD indirectly stimulates binding of Mcm10-ID to p180. Indeed, Mcm10-IDϩCTD binds both ssDNA and p180 189 -323 with 15-fold greater affinity than Mcm10-ID alone, suggesting that protein and DNA binding can be modified by domain interactions within Mcm10. However, anisotropy binding studies carried out with a mixture of Mcm10-ID and Mcm10-CTD did not enhance the binding affinity relative to either domain alone, and thus far we have been unable to observe a direct interaction between the ID and CTD. An alternate interpretation for the enhanced binding with the Mcm10-IDϩCTD construct is that a second binding site on the CTD provides an extended interaction surface for DNA, which results in a synergistic effect on binding similar to that observed for the multiple OB-fold binding motifs in RPA (72).
Mcm10 oligomerization provides a third mechanism for mediating DNA and p180 binding (Fig. 7C). Mcm10 has been reported to form dimeric and hexameric assemblies (25,73). We previously showed that Mcm10-NTD, which is predicted to contain a coiled-coil motif, forms a highly asymmetric dimer in solution (29). Dimerization of Mcm10 through the NTD would expose multiple IDϩCTD high affinity binding platforms for binding to DNA and/or pol ␣. The higher affinity of the IDϩCTD construct for both ssDNA and p180 189 -323 suggests that this is the preferred binding mode over the individual domains. Importantly, the similar affinities of Mcm10-IDϩCTD for pol ␣ and DNA provides a physical basis for simultaneous binding of ssDNA and pol ␣ by Mcm10. This condition also raises the possibility that a structural change would be necessary to facilitate Mcm10 release of pol ␣ during a molecular handoff to DNA. Previous studies suggest that phosphorylation (74) or ubiquitination (24) are likely candidates for altering Mcm10 binding affinities. Additional studies beyond the scope of this paper, including elucidating the structure of full-length Mcm10 and determining interaction partners, are required to fully understand how Mcm10 mediates critical interactions at the eukaryotic replication fork.