Mcm10 plays a key role in initiation and elongation of eukaryotic chromosomal DNA replication. As a first step to better understand the structure and function of vertebrate Mcm10, we have determined the structural architecture of Xenopus laevis Mcm10 (xMcm10) and characterized each domain biochemically. Limited proteolytic digestion of the full-length protein revealed N-terminal-, internal (ID)-, and C-terminal (CTD)-structured domains. Analytical ultracentrifugation revealed that xMcm10 self-associates and that the N-terminal domain forms homodimeric assemblies. DNA binding activity of xMcm10 was mapped to the ID and CTD, each of which binds to single-and double-stranded DNA with low micromolar affinity. The structural integrity of xMcm10-ID and CTD is dependent on the presence of bound zinc, which was experimentally verified by atomic absorption spectroscopy and proteolysis protection assays. The ID and CTD also bind independently to the N-terminal 323 residues of the p180 subunit of DNA polymerase ␣-primase. We propose that the modularity of the protein architecture, with discrete domains for dimerization and for binding to DNA and DNA polymerase ␣-primase, provides an effective means for coordinating the biochemical activities of Mcm10 within the replisome.Eukaryotic DNA replication is carried out by large multiprotein machines that coordinate DNA unwinding and synthesis at the replication fork. Initiation of replication involves ordered assembly of the replisome and local denaturation of duplex DNA at the origin followed by replisome activation. Screens for mutants defective in minichromosome maintenance (Mcm) 4 and DNA replication in yeast identified a number of factors essential for replication (1-4). Pre-replicative complexes composed of the origin recognition complex, Cdc6, Cdt1, and the hexameric Mcm2-7 helicase are assembled in G 1 (for review, see Ref. 5) and converted into active replication forks at the onset of S phase. Mcm10 loads onto chromatin after pre-replicative complex assembly (6, 7) and stimulates phosphorylation of Mcm2-7 by Dbf4-Cdc7 kinase (8). Once Mcm10 is present, Cdc45 and GINS are loaded onto chromatin (6, 9, 10) and form a Cdc45/Mcm2-7/GINS helicase complex (11-14). Cyclinand Dbf4-dependent kinases together with Sld2, Sld3, and Dpb11 in budding yeast (15, 16) stimulate origin unwinding, which is signified by recruitment of replication protein A to singlestranded DNA (17, 18). Mcm10, Cdc45, and replication protein A facilitate subsequent loading of DNA polymerase ␣-primase (pol ␣) onto chromatin (7,9,19,20). The association of proliferating cell nuclear antigen, RFC, and replicative DNA polymerases ␦ and ⑀ with the origin completes the replisome (for review, see Ref. 21).A number of interactions have been observed between Mcm10 and proteins found in the pre-replicative complexes and at the replication fork. Mcm10 is a component of active replication complexes in Xenopus and budding yeast (12,14) and is associated with chromatin throughout S-phase (7). Mcm10 interacts genetically w...

Summary Mcm10 is an essential eukaryotic DNA replication protein required for assembly and progression of the replication fork. The highly conserved internal domain (Mcm10-ID) has been shown to physically interact with single-stranded (ss) DNA, DNA polymerase α, and PCNA. The crystal structure of Xenopus laevis Mcm10-ID presented here reveals a novel DNA binding architecture composed of an OB-fold followed in tandem by a variant and highly basic zinc finger. NMR chemical shift perturbation and mutational studies of DNA binding activity in vitro reveal how Mcm10 uses this unique surface to engage ssDNA. Corresponding mutations in Saccharomyces cerevisiae result in increased sensitivity to replication stress, demonstrating the functional importance of DNA binding by this region of Mcm10 to replication. In addition, mapping Mcm10 mutations known to disrupt PCNA, pol α, and DNA interactions onto the crystal structure provides insight into how Mcm10 may coordinate protein and DNA binding within the replisome.

DNA replication requires priming of DNA templates by enzymes known as primases. Although DNA primase structures are available from archaea and bacteria, the mechanism of DNA priming in higher eukaryotes remains poorly understood in large part due to the absence of the structure of the unique, highly conserved C-terminal regulatory domain of the large subunit (p58C). Here, we present the structure of this domain determined to 1.7-Å resolution by X-ray crystallography. The p58C structure reveals a novel arrangement of an evolutionarily conserved 4Fe-4S cluster buried deeply within the protein core and is not similar to any known protein structure. Analysis of the binding of DNA to p58C by fluorescence anisotropy measurements revealed a strong preference for ss/ dsDNA junction substrates. This approach was combined with site-directed mutagenesis to confirm that the binding of DNA occurs to a distinctively basic surface on p58C. A specific interaction of p58C with the C-terminal domain of the intermediate subunit of replication protein A (RPA32C) was identified and characterized by isothermal titration calorimetry and NMR. Restraints from NMR experiments were used to drive computational docking of the two domains and generate a model of the p58C-RPA32C complex. Together, our results explain functional defects in human DNA primase mutants and provide insights into primosome loading on RPA-coated ssDNA and regulation of primase activity.he replisome is a dynamic assembly of proteins that carries out eukaryotic DNA replication. The first step in the initiation of DNA replication on both the leading and lagging strand ssDNA is the synthesis of a short (8-10 nucleotide) RNA primer by a polymerase termed a DNA primase (1-3). DNA primases are unique because they are the sole polymerases capable of de novo synthesis on a ssDNA template. Although integral to the replisome, the activity of DNA primase is not limited to DNA replication; primase is also an essential component of the DNA damage response and plays a role in telomere maintenance (4, 5).Although all primases serve a common function, their architectures and modes of interaction with other protein factors vary among species (6, 7). The human DNA primase is part of the heterotetrameric polymerase α-primase (pol-prim) complex, which generates short (∼30 nucleotide) stretches of DNA primed with chimeric RNA-DNA hybrids that are handed off to the primary replicative DNA polymerases. The p48 and p58 subunits of human pol-prim form the DNA primase heterodimer. Although the p48 subunit contains the RNA polymerase catalytic active site, both subunits are required for synthesis of short RNA primers (8, 9). The p58 subunit is proposed to play various roles, including stabilizing the p48 subunit, recognizing the DNA template, and promoting initiation and elongation of primers (9, 10).Considerable knowledge about the molecular mechanism of DNA priming has been obtained from structural studies of archaeal and bacterial primases (11, 12). In contrast, the mechanism of DNA priming ...

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 ␣ (pol ␣), 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 ␣. However, the mechanism by which Mcm10 interacts with pol ␣ 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-ID⅐ssDNA 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 ␣ within the replication fork.

The cytoplasmic domain of the anion exchange protein (cdb3) serves as a critical organizing center for protein-protein interactions that stabilize the erythrocyte membrane. The structure of the central core of cdb3, determined by X-ray crystallography from crystals grown at pH 4.8, revealed a compact dimer for residues 55-356 and unresolved N- and C-termini on each monomer [Zhang et al. (2000) Blood 96, 2925-2933]. Given that previous studies had suggested a highly asymmetric structure for cdb3 and that pH dependent structural transitions of cdb3 have been reported, the structure of cdb3 in solution at neutral pH was investigated via site-directed spin labeling in combination with conventional electron paramagnetic resonance (EPR) and double electron electron resonance (DEER) spectroscopies. These studies show that the structure of the central compact dimer (residues 55-356) is indistinguishable from the crystal structure determined at pH 4.8. N-Terminal residues 1-54 and C-terminal residues 357-379 are dynamically disordered and show no indications of stable secondary structure. These results establish a structural model for cdb3 in solution at neutral pH which represents an important next step in characterizing structural details of the protein-protein interactions that stabilize the erythrocyte membrane.

While type determination on bullets has been performed for over a century, type determination on cartridge cases is often overlooked. Presented here is an example of type determination of ejector marks on cartridge cases from Glock and Smith& Wesson Sigma series pistols using Naïve Bayes and Random Forest classification methods. The shapes of ejector marks were captured from images of test-fired cartridge cases and subjected to multivariate analysis. Naïve Bayes and Random Forest methods were used to assign the ejector shapes to the correct class of firearm with success rates as high as 98%. This method is easily implemented with equipment already available in crime laboratories and can serve as an investigative lead in the form of a list of firearms that could have fired the evidence. Paired with the FBI's General Rifling Characteristics (GRC) database, this could be an invaluable resource for firearm evidence at crime scenes.

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