Abstract
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 alpha (pol alpha), 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 alpha. However, the mechanism by which Mcm10 interacts with pol alpha 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 alpha within the replication fork.
Highlights
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 oligosaccharide binding (OB)-fold cleft of Mcm10
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)
Calorimetric titration of Mcm10-ID with p180189–323 (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
Summary
Protein Expression and Purification—Mcm10-ID was prepared as described previously [31]. 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. Chemical shift perturbation data were collected by titrating 1.8 mM unlabeled p180189 –323 into 400 M 15N-labeled Mcm10-ID in 20 mM Tris-d11, pH 7.0, 75 mM NaCl, and 5% D2O. For binding measurements, unlabeled Mcm protein was added over a concentration range of 0.1–50 M to solutions containing either 50 nM fluorescein-p180 fragment or 50 nM fluoresceinDNA. Under these conditions, both fluorescein-labeled molecules are Ͼ80% saturated (Fig. 2B and Ref. 31). 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 Ka
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