DNA primase synthesizes short RNA primers that are required to initiate DNA synthesis on the parental template strands during DNA replication. Eukaryotic primase contains two subunits, p48 and p58, and is normally tightly associated with DNA polymerase ␣. Despite the fundamental importance of primase in DNA replication, structural data on eukaryotic DNA primase are lacking. The p48/p58 dimer was subjected to limited proteolysis, which produced two stable structural domains: one containing the bulk of p48 and the other corresponding to the C-terminal fragment of p58. These domains were identified by mass spectrometry and N-terminal sequencing. The C-terminal p58 domain (p58C) was expressed, purified, and characterized. CD and NMR spectroscopy experiments demonstrated that p58C forms a well folded structure. The protein has a distinctive brownish color, and evidence from inductively coupled plasma mass spectrometry, UV-visible spectrophotometry, and EPR spectroscopy revealed characteristics consistent with the presence of a [4Fe-4S] high potential iron protein cluster. Four putative cysteine ligands were identified using a multiple sequence alignment, and substitution of just one was sufficient to cause loss of the iron-sulfur cluster and a reduction in primase enzymatic activity relative to the wildtype protein. The discovery of an iron-sulfur cluster in DNA primase that contributes to enzymatic activity provides the first suggestion that the DNA replication machinery may have redox-sensitive activities. Our results offer new horizons in which to investigate the function of high potential [4Fe-4S] clusters in DNA-processing machinery.DNA polymerase ␣-primase (pol-prim) 2 associates with eukaryotic replication forks in S-phase during the initiation of DNA replication (1, 2). pol-prim synthesizes a chimeric RNA-DNA primer of ϳ30 nucleotides that is then extended by more processive DNA polymerases that synthesize the leading and lagging strands. pol-prim is composed of four subunits (p180, p68, p58, and p48). The p180 subunit has the DNA polymerase catalytic activity and binds to both the p68 and p58 subunits. The p68 subunit has a regulatory function that is not completely understood. It is required for initiation of yeast chromosomal replication (3, 4) and cell-free SV40 DNA replication (5). In addition, phosphorylation of p68 alters the activity of polprim in SV40 replication (6 -9).The two smallest subunits, p48 and p58, together function as the DNA primase by creating an RNA primer of 7-10 nucleotides (10, 11). The p48 subunit contains the catalytic site (12). The p58 subunit stabilizes p48 and participates in initiation, elongation, and "counting" the ribonucleotides polymerized (13). Interestingly, p58 is also involved in transferring the RNA strand directly into the active site of the associated p180 subunit, which extends the growing nucleotide with dNTPs to complete the formation of the RNA-DNA primer (1, 14, 15). Knowledge of the molecular basis for regulation of the length of RNA portion of the primer an...

We report that during activation of the simian virus 40 (SV40) pre-replication complex, SV40 T antigen (Tag) helicase actively loads replication protein A (RPA) on emerging single-stranded DNA (ssDNA). This novel loading process requires physical interaction of Tag origin DNA-binding domain (OBD) with the RPA high-affinity ssDNA-binding domains (RPA70AB). Heteronuclear NMR chemical shift mapping revealed that Tag-OBD binds to RPA70AB at a site distal from the ssDNA-binding sites and that RPA70AB, Tag-OBD, and an 8-nucleotide ssDNA form a stable ternary complex. Intact RPA and Tag also interact stably in the presence of an 8-mer, but Tag dissociates from the complex when RPA binds to longer oligonucleotides. Together, our results imply that an allosteric change in RPA quaternary structure completes the loading reaction. A mechanistic model is proposed in which the ternary complex is a key intermediate that directly couples origin DNA unwinding to RPA loading on emerging ssDNA.

The topology of most experimentally determined protein domains is defined by the relative arrangement of secondary structure elements, i.e. α-helices and β-strands, which make up 50–70% of the sequence. Pairing of β-strands defines the topology of β-sheets. The packing of side chains between α-helices and β-sheets defines the majority of the protein core. Often, limited experimental datasets restrain the position of secondary structure elements while lacking detail with respect to loop or side chain conformation. At the same time the regular structure and reduced flexibility of secondary structure elements make these interactions more predictable when compared to flexible loops and side chains. To determine the topology of the protein in such settings, we introduce a tailored knowledge-based energy function that evaluates arrangement of secondary structure elements only. Based on the amino acid Cβ atom coordinates within secondary structure elements, potentials for amino acid pair distance, amino acid environment, secondary structure element packing, β-strand pairing, loop length, radius of gyration, contact order and secondary structure prediction agreement are defined. Separate penalty functions exclude conformations with clashes between amino acids or secondary structure elements and loops that cannot be closed. Each individual term discriminates for native-like protein structures. The composite potential significantly enriches for native-like models in three different databases of 10,000–12,000 protein models in 80–94% of the cases. The corresponding application, “BCL::ScoreProtein,” is available at www.meilerlab.org.

Computational de novo protein structure prediction is limited to small proteins of simple topology. The present work explores an approach to extend beyond the current limitations through assembling protein topologies from idealized α-helices and β-strands. The algorithm performs a Monte Carlo Metropolis simulated annealing folding simulation. It optimizes a knowledge-based potential that analyzes radius of gyration, β-strand pairing, secondary structure element (SSE) packing, amino acid pair distance, amino acid environment, contact order, secondary structure prediction agreement and loop closure. Discontinuation of the protein chain favors sampling of non-local contacts and thereby creation of complex protein topologies. The folding simulation is accelerated through exclusion of flexible loop regions further reducing the size of the conformational search space. The algorithm is benchmarked on 66 proteins with lengths between 83 and 293 amino acids. For 61 out of these proteins, the best SSE-only models obtained have an RMSD100 below 8.0 Å and recover more than 20% of the native contacts. The algorithm assembles protein topologies with up to 215 residues and a relative contact order of 0.46. The method is tailored to be used in conjunction with low-resolution or sparse experimental data sets which often provide restraints for regions of defined secondary structure.

Modular proteins with multiple domains tethered by flexible linkers have variable global archiectures. Using the eukaryotic ssDNA binding protein, Replication Protein A (RPA), we demonstrate that NMR spectroscopy is a powerful tool to characterize the remodeling of architecture in different functional states. The first direct evidence is obtained for the remodeling of RPA upon binding ssDNA, including an alteration in the availability of the RPA32N domain that may help explain its damage-dependent phosphorylation.The progression of DNA replication and repair requires the coordinated action of dynamic, multi-protein assemblies. We have previously proposed a critical role for proteins composed of multiple, flexibly attached domains in facilitating the action of these dynamic complexes 1 . Because these proteins can undergo intra-and inter-domain rearrangements, they are able to interact optimally with the ever-changing substrate landscape present during DNA processing. RPA is a prototypical modular multi-domain DNA processing protein with flexible linkers of various lengths (Figure 1). The trimer core is a compact assembly of three OB-fold domains (RPA70C/32D/14) to which is appended the disordered RPA32N functional domain, the RPA32C winged-helix domain, and the tandem RPA70AB and the RPA70N OB-fold NMR spectroscopy in solution is a powerful tool for characterizing proteins under conditions that preserve intrinsic dynamic properties. The advent of TROSY, CRINEPT and related experimental approaches 3 has vastly increased the upper limit of molecular masses accessible to study by NMR. Examples range from the globular malate synthase (82 kDa) to the oligomeric GroEL-GroES complex (872 kDa) to highly flexible domains from the ribosome (>2.5 MDa) 4 . In the case of RPA (116 kDa) and many other multi-domain proteins, modularity and interdomain flexibility are the critical properties that enable characterization of dynamic architectures by NMR.To illustrate the analytical framework, results are presented first for RPA70NAB (M r 45.8 kDa), which has an asymmetric arrangement with a 70-residue N-A linker and a 10-residue A-B linker (Figure 1). The 15 N-1 H TROSY-HSQC spectrum of 15 N-enriched RPA70NAB reveals the presence of over 370 of the 400 expected signals from 422 residues (Figure 2). The signals from each of the three domains appear in positions remarkably similar to those in NMR spectra of the three isolated domains ( Figure S1). Thus, all three domains are structurally independent and resonance assignments can be transferred from the isolated domains to RPA70NAB 5 . NMR is highly sensitive to differences in the degree of inter-domain flexibility; the signals from the A and B domains are substantially weaker than the signals from the N domain, even though all three domains are approximately the same mass ( Figure 2). The differences arise from the fact that although the A and B domains are structurally independent, the short A-B tether partially restricts their motions, whereas the much longer N-A tether e...

Summary Membrane protein structure determination remains a challenging endeavor. Computational methods that predict membrane protein structure from sequence can potentially aid structure determination for such difficult target proteins. The de novo protein structure prediction method, BCL::Fold, rapidly assembles secondary structure elements into 3-dimensional models. Here we describe modifications to the algorithm, named BCL::MP-Fold, in order to simulate membrane protein folding. Models are built into a static membrane object and are evaluated using a knowledge-based energy potential, which has been modified to account for the membrane environment. Additionally, a new symmetry folding mode allows for the prediction of obligate homomultimers, a common property amongst membrane proteins. In a benchmark test of 40 proteins of known structure, the method samples the correct topology in 34 cases. This demonstrates that the algorithm can accurately predict protein topology without the need for large multiple sequence alignments, homologous template structures, or experimental restraints.

For many membrane proteins, the determination of their topology remains a challenge for methods like X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. Electron paramagnetic resonance (EPR) spectroscopy has evolved as an alternative technique to study structure and dynamics of membrane proteins. The present study demonstrates the feasibility of membrane protein topology determination using limited EPR distance and accessibility measurements. The BCL::MP-Fold algorithm assembles secondary structure elements (SSEs) in the membrane using a Monte Carlo Metropolis (MCM) approach. Sampled models are evaluated using knowledge-based potential functions and agreement with the EPR data and a knowledge-based energy function. Twenty-nine membrane proteins of up to 696 residues are used to test the algorithm. The protein-size-normalized root-mean-square-deviation (RMSD100) value of the most accurate model is better than 8 Å for twenty-seven, better than 6 Å for twenty-two, and better than 4 Å for fifteen out of twenty-nine proteins, demonstrating the algorithm’s ability to sample the native topology. The average enrichment could be improved from 1.3 to 2.5, showing the improved discrimination power by using EPR data.

DNA polymerase ␣-primase (pol-prim) plays a central role in DNA replication in higher eukaryotes, initiating synthesis on both leading and lagging strand single-stranded DNA templates. Pol-prim consists of a primase heterodimer that synthesizes RNA primers, a DNA polymerase that extends them, and a fourth subunit, p68 (also termed B-subunit), that is thought to regulate the complex. Although significant knowledge about single-subunit primases of prokaryotes has accumulated, the functions and regulation of pol-prim remain poorly understood. In the SV40 replication model, the p68 subunit is required for primosome activity and binds directly to the hexameric viral helicase T antigen, suggesting a functional link between T antigen-p68 interaction and primosome activity. To explore this link, we first mapped the interacting regions of the two proteins and discovered a previously unrecognized N-terminal globular domain of p68 (p68N) that physically interacts with the T antigen helicase domain. NMR spectroscopy was used to determine the solution structure of p68N and map its interface with the T antigen helicase domain. Structure-guided mutagenesis of p68 residues in the interface diminished T antigen-p68 interaction, confirming the interaction site. SV40 primosome activity of corresponding pol-prim mutants decreased in proportion to the reduction in p68N-T antigen affinity, confirming that p68-T antigen interaction is vital for primosome function. A model is presented for how this interaction regulates SV40 primosome activity, and the implications of our findings are discussed in regard to the molecular mechanisms of eukaryotic DNA replication initiation.De novo DNA replication begins with RNA primer synthesis on a DNA template, followed by primer extension and processive DNA synthesis. In prokaryotes, a primosome couples activity of primase with parental DNA unwinding by a hexameric helicase and a single-stranded DNA (ssDNA) 5 -binding protein, largely through dynamic physical associations among the three proteins (1, 2). In eukaryotes, the core of the primosome is the DNA polymerase ␣-primase complex (pol-prim), which catalyzes both RNA primer synthesis and extension into RNA-DNA primers (3). Pol-prim initiates synthesis of both leading and lagging strands at eukaryotic replication origins and is required during elongation to initiate synthesis of each Okazaki fragment on the lagging strand. Pol-prim also plays a vital role in telomere maintenance and intra-S phase checkpoint activity. However, the mechanisms that regulate recruitment and activity of pol-prim in these various settings remain poorly understood.Pol-prim is a complex of four subunits. Its primase subunits (p48 and p58) initially synthesize an 8 -12-nucleotide RNA primer, which is then shifted internally to the active site of the associated p180 DNA polymerase subunit for extension into a 30 -35-nucleotide RNA-DNA primer (4, 5). The fourth subunit of the pol-prim complex, known as p68 or B-subunit, lacks enzyme activity but is essential for S phase entr...