Solution NMR Structure of the C-terminal DNA Binding Domain of Mcm10 Reveals a Conserved MCM Motif

The eukaryotic DNA replication protein Mcm10 associates with chromatin in early S-phase and is required for assembly and function of the replication fork protein machinery. Xenopus laevis (X) Mcm10 binds DNA via a highly conserved internal domain (ID) and a C-terminal domain (CTD) that is unique to higher eukaryotes. Although the structural basis of the interactions of the ID with DNA and polymerase α is known, little information is available for the CTD. We have identified the minimal DNA binding region of the XMcm10-CTD and determined its three-dimensional structure by solution NMR. The CTD contains a globular domain composed of two zinc binding motifs. NMR chemical shift perturbation and mutational analysis show that ssDNA binds only to the N-terminal (CCCH-type) zinc motif, whose structure is unique to Mcm10. The second (CCCC-type) zinc motif is not involved in DNA binding. However, it is structurally similar to the CCCC zinc ribbon in the N-terminal oligomerization domain of eukaryotic and archaeal MCM helicases. NMR analysis of a construct spanning both the ID and CTD reveals that the two DNA binding domains are structurally independent in solution, supporting a modular architecture for vertebrate Mcm10. Our results provide insight in the action of Mcm10 in the replisome and support a model in which it serves as a central scaffold through coupling of interactions with partner proteins and the DNA.     

The structural basis for substrate specificity in DNA topoisomerase IV

Most bacteria possess two type IIA topoisomerases, DNA gyrase and topo IV, that together help manage chromosome integrity and topology. Gyrase primarily introduces negative supercoils into DNA, an activity mediated by the C-terminal domain of its DNA binding subunit (GyrA). Although closely related to gyrase, topo IV preferentially decatenates DNA and relaxes positive supercoils. Here we report the structure of the full-length Escherichia coli ParC dimer at 3.0 A resolution. The N-terminal DNA binding region of ParC is highly similar to that of GyrA, but the ParC dimer adopts a markedly different conformation. The C-terminal domain (CTD) of ParC is revealed to be a degenerate form of the homologous GyrA CTD, and is anchored to the top of the N-terminal domains in a configuration different from that thought to occur in gyrase. Biochemical assays show that the ParC CTD controls the substrate specificity of topo IV, likely by capturing DNA segments of certain crossover geometries. This work delineates strong mechanistic parallels between topo IV and gyrase, while explaining how structural differences between the two enzyme families have led to distinct activity profiles. These findings in turn explain how the structures and functions of bacterial type IIA topoisomerases have evolved to meet specific needs of different bacterial families for the control of chromosome superstructure.     

Primer utilization by DNA polymerase alpha-primase is influenced by its interaction with Mcm10p

Models of DNA replication in yeast and Xenopus suggest that Mcm10p is required to generate the pre-initiation complex as well as progression of the replication fork during the elongation of DNA chains. In this report, we show that the Schizosaccharomyces pombe Mcm10p/Cdc23p binds to the S. pombe DNA polymerase (pol) alpha-primase complex in vitro by interacting specifically with the catalytic p180 subunit and stimulates DNA synthesis catalyzed by the pol alpha-primase complex with various primed DNA templates. We investigated the mechanism by which Mcm10p activates the polymerase activity of the pol alpha-primase complex by generating truncated derivatives of the full-length 593-amino acid Mcm10p. Their ability to stimulate pol alpha polymerase activity and bind to single-stranded DNA and to pol alpha were compared. Concomitant with increased deletion of the N-terminal region (from amino acids 95 to 415), Mcm10p derivatives lost their ability to stimulate pol alpha polymerase activity and bind to single-stranded DNA. Truncated derivatives of Mcm10p containing amino acids 1-416 retained the pol alpha binding activity, whereas the C terminus, amino acids 496-593, did not. These results demonstrate that both the single-stranded DNA binding and the pol alpha binding properties of Mcm10p play important roles in the activation. In accord with these findings, Mcm10p facilitated the binding of pol alpha-primase complex to primed DNA and formed a stable complex with pol alpha-primase on primed templates. A mutant that failed to activate or bind to DNA and pol alpha, was not observed in this complex. We suggest that the interaction of Mcm10p with the pol alpha-primase complex, its binding to single-stranded DNA, and its activation of the polymerase complex together contribute to its role in the elongation phase of DNA replication.     

LAST motifs and SMART domains in gene 32 protein: an unfolding story of autoregulation?

Bacteriophage T4 gene 32 protein is a classical single strand-specific DNA binding protein. It is a single polypeptide chain of 301 amino acid residues that consists of three structural domains, each of which has a binding function. The N-terminal domain is involved in homotypic protein-protein interaction (the basis of binding cooperativity), the core domain binds single strands directly, and the C-terminal domain has a role in heterotypic protein-protein association. The three domains have traditionally been thought to be independent of each other. However, the observation of a striking repetition of a basic, polar sequence (the "LAST" Motif), seen in both the N-terminal and core domains, suggests a linkage between these domains. Moreover, the C-domain and adjoining portion (flap) of the core are highly acidic, and are potential mimics of single-stranded DNA. With these observations, I construct a model in which this flap is associated with the ssDNA binding site in the absence of DNA, and upon cooperative protein binding to DNA, the flap now associates with the N-terminal domain of the adjacent DNA-bound protein. The flap thus acts as a gate, which might slow the binding of the protein to DNA. This could lead to the regulation of the protein's various interactions with other proteins, as well as affect its ability to lower DNA melting temperature.     

Physical Interactions between Mcm10, DNA, and DNA Polymerase ��

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 (5–8). Origin unwinding is followed by loading of RPA,³ And-1/Ctf4, and pol α 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 δ and ϵ 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, 17–19). 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 α and helps to maintain its association with chromatin (16, 26, 27). This is a critical interaction during replication because pol α is the only enzyme in eukaryotic cells that is capable of initiating DNA synthesis de novo. Indeed, Mcm10 stimulates the polymerase activity of pol α 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 α (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 composed 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 α has been identified in the sequence of Saccharomyces cerevisiae Mcm10-ID (16, 26).DNA pol α-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 α 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 ∼30 nucleotides, replication factor C binds to the nascent strand to displace pol α and loads PCNA with pols δ and ϵ (for review, see Ref. 38).The interaction between Mcm10 and pol α 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 α 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 α binding in the presence of ssDNA. Collectively, these data demonstrate a shared binding site for DNA and pol α in the OB-fold cleft of Mcm10-ID, with a preference for ssDNA over pol α. In addition, we have mapped the Mcm10-ID binding site on pol α to a 24-residue segment of the N-terminal domain of p180. Based on these results, we propose Mcm10 helps to recruit pol α to origins of replication by a molecular hand-off mechanism.     

The BARD1 C-terminal domain structure and interactions with polyadenylation factor CstF-50

The BARD1 N-terminal RING domain binds BRCA1 while the BARD1 C-terminal ankyrin and tandem BRCT repeat domains bind CstF-50 to modulate mRNA processing and RNAP II stability in response to DNA damage. Here we characterize the BARD1 structural biochemistry responsible for CstF-50 binding. The crystal structure of the BARD1 BRCT domain uncovers a degenerate phosphopeptide binding pocket lacking the key arginine required for phosphopeptide interactions in other BRCT proteins. Small angle X-ray scattering together with limited proteolysis results indicates that ankyrin and BRCT domains are linked by a flexible tether and do not adopt a fixed orientation relative to one another. Protein pull-down experiments utilizing a series of purified BARD1 deletion mutants indicate that interactions between the CstF-50 WD-40 domain and BARD1 involve the ankyrin-BRCT linker but do not require ankyrin or BRCT domains. The structural plasticity imparted by the ANK-BRCT linker helps to explain the regulated assembly of different protein BARD1 complexes with distinct functions in DNA damage signaling including BARD1-dependent induction of apoptosis plus p53 stabilization and interactions. BARD1 architecture and plasticity imparted by the ANK-BRCT linker are suitable to allow the BARD1 C-terminus to act as a hub with multiple binding sites to integrate diverse DNA damage signals directly to RNA polymerase.     

Role of the insertion domain and the zinc-finger motif of Escherichia coli UvrA in damage recognition and ATP hydrolysis

UvrA is the initial DNA damage-sensing protein in bacterial nucleotide excision repair. Each protomer of the UvrA dimer contains two ATPase domains, that belong to the family of ATP-binding cassette domains. Three structural domains are inserted in these ATPase domains: the insertion domain (ID) and UvrB binding domain (in ATP domain I) and the zinc-finger motif (in ATP domain II). In this paper we analyze the function of the ID and the zinc finger motif in damage specific binding of Escherichia coli UvrA. We show that the ID is not essential for damage discrimination, but it does stabilize UvrA on the DNA, most likely by forming a clamp around the DNA helix. We present evidence that two conserved arginine residues in the ID contact the phosphate backbone of the DNA, leading to strand separation after the ATPase-driven movement of the ID's. Remarkably, deletion of the ID generated a phenotype in which UV-survival strongly depends on the presence of photolyase, indicating that UvrA and photolyase form a ternary complex on a CPD-lesion. The zinc-finger motif is shown to be important for the transfer of the damage recognition signal to the ATPase of UvrA. In the absence of this domain the coupling between DNA binding and ATP hydrolysis is completely lost. Mutation of the phenylalanine residue in the tip of the zinc-finger domain resulted in a protein in which the ATPase was already triggered when binding to an undamaged site. As the zinc-finger motif is connected to the DNA binding regions on the surface of UvrA, this strongly suggests that damage-specific binding to these regions results in a rearrangement of the zinc-finger motif, which in its turn activates the ATPase. We present a model how damage recognition is transmitted to activate ATP hydrolysis in ATP binding domain I of the protein.     

Multifaceted recognition of vertebrate Rev1 by translesion polymerases ζ and κ

Translesion synthesis is a fundamental biological process that enables DNA replication across lesion sites to ensure timely duplication of genetic information at the cost of replication fidelity, and it is implicated in development of cancer drug resistance after chemotherapy. The eukaryotic Y-family polymerase Rev1 is an essential scaffolding protein in translesion synthesis. Its C-terminal domain (CTD), which interacts with translesion polymerase ζ through the Rev7 subunit and with polymerases κ, ι, and η in vertebrates through the Rev1-interacting region (RIR), is absolutely required for function. We report the first solution structures of the mouse Rev1 CTD and its complex with the Pol κ RIR, revealing an atypical four-helix bundle. Using yeast two-hybrid assays, we have identified a Rev7-binding surface centered at the α2-α3 loop and N-terminal half of α3 of the Rev1 CTD. Binding of the mouse Pol κ RIR to the Rev1 CTD induces folding of the disordered RIR peptide into a three-turn α-helix, with the helix stabilized by an N-terminal cap. RIR binding also induces folding of a disordered N-terminal loop of the Rev1 CTD into a β-hairpin that projects over the shallow α1-α2 surface and creates a deep hydrophobic cavity to interact with the essential FF residues juxtaposed on the same side of the RIR helix. Our combined structural and biochemical studies reveal two distinct surfaces of the Rev1 CTD that separately mediate the assembly of extension and insertion translesion polymerase complexes and provide a molecular framework for developing novel cancer therapeutics to inhibit translesion synthesis.     

DNA binding properties of protein TrwA, a possible structural variant of the Arc repressor superfamily

Conjugative DNA processing of plasmid R388 requires the concerted action of two proteins, the relaxase-helicase TrwC and the relaxase enhancer TrwA. TrwA can be aligned with DNA binding proteins belonging to the ribbon-helix-helix (RHH) protein family. To further analyse TrwA function, the structural domains of the protein have been identified and dissected by limited proteolysis. Two stable domains were found that resulted to be, according to DNA binding experiments and oligomerization analysis, an N-terminal DNA binding domain and a C-terminal tetramerization domain. Using the three-dimensional structure of the Arc repressor as a guide, it was possible to model TrwA DNA binding site with atomic detail. As a result, TrwA polar amino acids Q8, R10 and S12, contained in the polar face of a putative N-terminal beta-strand, were found to be directly involved in DNA binding, in a manner analogous to RHH proteins. In this respect, TrwA seemed to be a new member of the RHH family. However, secondary structure analyses underscored the existence of a substantial difference in the architecture of the TrwA-oriT complex when compared to the Arc repressor-operator complex.     

Functional conservation of beta-hairpin DNA binding domains in the Mcm protein of Methanobacterium thermoautotrophicum and the Mcm5 protein of Saccharomyces cerevisiae

Mcm proteins are an important family of evolutionarily conserved helicases required for DNA replication in eukaryotes. The eukaryotic Mcm complex consists of six paralogs that form a heterohexameric ring. Because the intact Mcm2-7 hexamer is inactive in vitro, it has been difficult to determine the precise function of the different subunits. The solved atomic structure of an archaeal minichromosome maintenance (MCM) homolog provides insight into the function of eukaryotic Mcm proteins. The N-terminal positively charged central channel in the archaeal molecule consists of beta-hairpin domains essential for DNA binding in vitro. Eukaryotic Mcm proteins also have beta-hairpin domains, but their function is unknown. With the archaeal atomic structure as a guide, yeast molecular genetics was used to query the function of the beta-hairpin domains in vivo. A yeast mcm5 mutant with beta-hairpin mutations displays defects in the G1/S transition of the cell cycle, the initiation phase of DNA replication, and in the binding of the entire Mcm2-7 complex to replication origins. A similar mcm4 mutation is synthetically lethal with the mcm5 mutation. Therefore, in addition to its known regulatory role, Mcm5 protein has a positive role in origin binding, which requires coordination by all six Mcm2-7 subunits in the hexamer.