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.     

Zinc stoichiometry of yeast RNA polymerase II and characterization of mutations in the zinc-binding domain of the largest subunit

Atomic absorption spectroscopy demonstrated that highly purified RNA polymerase II from the yeast Saccharomyces cerevisiae binds seven zinc ions. This number agrees with the number of potential zinc-binding sites among the 12 different subunits of the enzyme and with our observation that the ninth largest subunit alone is able to bind two zinc ions. The zinc-binding motif in the largest subunit of the enzyme was investigated using mutagenic analysis. Altering any one of the six conserved residues in the zinc-binding motif conferred either a lethal or conditional phenotype, and zinc blot analysis indicated that mutant forms of the domain had a 2-fold reduction in zinc affinity. Mutations in the zinc-binding domain reduced RNA polymerase II activity in cell-free extracts, even though protein blot analysis indicated that the mutant subunit was present in excess of wild-type levels. Purification of one mutant RNA polymerase revealed a subunit profile that was wild-type like with the exception of two subunits not required for core enzyme activity (Rpb4p and Rpb7p), which were missing. Core activity of the mutant enzyme was reduced 20-fold. We conclude that mutations in the zinc-binding domain can reduce core activity without altering the association of any of the subunits required for this activity.     

A zinc-binding region in Vif binds Cul5 and determines cullin selection

Human immunodeficiency virus-1 (HIV-1) Vif overcomes the anti-viral activity of APOBEC3G by targeting it for ubiquitination via a Cullin 5-ElonginB-ElonginC (Cul5-EloBC) E3 ligase. Vif associates with Cul5-EloBC through a BC-box motif that binds EloC, but the mechanism by which Vif selectively recruits Cul5 is poorly understood. Here we report that a region of Vif (residues 100-142) upstream of the BC-box binds selectively to Cul5 in the absence of EloC. This region contains a zinc coordination site HX5CX17-18CX3-5H (HCCH), with His/Cys residues at positions 108, 114, 133, and 139 coordinating one zinc ion. The HCCH zinc coordination site, which is conserved among primate lentivirus Vif proteins, does not correspond to any known class of zinc-binding motif. Mutations of His/Cys residues in the HCCH motif impair zinc coordination, Cul5 binding, and APOBEC3G degradation. Mutations of conserved hydrophobic residues (Ile-120, Ala-123, and Leu-124) located between the two Cys residues in the HCCH motif disrupt binding of the zinc-coordinating region to Cul5 and inhibit APOBEC3G degradation. The Vif binding site maps to the first cullin repeat in the N terminus of Cul5. These data suggest that the zinc-binding region in Vif is a novel cullin interaction domain that mediates selective binding to Cul5. We propose that the HCCH zinc-binding motif facilitates Vif-Cul5 binding by playing a structural role in positioning hydrophobic residues for direct contact with Cul5.     

REC, a new member of the MCM-related protein family, is required for meiotic recombination in Drosophila

rec mutations result in an extremely low level of recombination and a high frequency of primary non-disjunction in the female meiosis of Drosophila melanogaster. Here we demonstrate that the rec gene encodes a novel protein related to the mini-chromosome maintenance (MCM) proteins. Six MCM proteins (MCM2-7) are conserved in eukaryotic genomes, and they function as heterohexamers in the initiation and progression of mitotic DNA replication. Three rec alleles, rec(1), rec(2) and rec (3), were found to possess mutations within this gene, and P element-mediated germline transformation with a wild-type rec cDNA fully rescued the rec mutant phenotypes. The 885 amino acid REC protein has an MCM domain in the middle of its sequence and, like MCM2, 4, 6 and 7, REC contains a putative Zn-finger motif. Phylogenetic analyses revealed that REC is distantly related to the six conserved MCM proteins. Database searches reveal that there are candidates for orthologs of REC in other higher eukaryotes, including human. We addressed whether rec is involved in DNA repair in the mitotic division after the DNA damage caused by methylmethane sulfonate (MMS) or by X-rays. These analyses suggest that the rec gene has no, or only a minor, role in DNA repair and recombination in somatic cells.     

Mutational analysis of conserved aspartic acid residues in the <Emphasis Type="Italic">Methanothermobacter thermautotrophicus</Emphasis> MCM helicase

Minichromosome maintenance (MCM) helicases are thought to function as the replicative helicases in archaea and eukarya, unwinding the duplex DNA in the front of the replication fork. The archaeal MCM helicase can be divided into three parts, the N-terminal, catalytic, and C-terminal regions. The N-terminal part of the protein is divided into three domains, A, B, and C, and was shown to be involved in protein multimerization and binding to single- and double-stranded DNA. Two Asp residues found in domain C are conserved among MCM proteins from different archaea. These residues are located in a loop at the interface with domain A. Mutations of these residues in the Methanothermobacter thermautotrophicus MCM protein, Asp202 and Asp203, to Asn result in a significant reduction in the ability of the enzyme to bind DNA and in lower thermal stability. However, the mutant proteins retained helicase and ATPase activities. Further investigation of the DNA binding revealed that the presence of ATP rescues the DNA binding deficiencies by these mutant proteins. Possible roles of these conserved residues in MCM function are discussed.     

Interactions between the archaeal Cdc6 and MCM proteins modulate their biochemical properties

The origin recognition complex, Cdc6 and the minichromosome maintenance (MCM) complex play essential roles in the initiation of eukaryotic DNA replication. Homologs of these proteins may play similar roles in archaeal replication initiation. While the interactions among the eukaryotic initiation proteins are well documented, the protein–protein interactions between the archaeal proteins have not yet been determined. Here, an extensive structural and functional analysis of the interactions between the Methanothermobacter thermautotrophicus MCM and the two Cdc6 proteins (Cdc6-1 and -2) identified in the organism is described. The main contact between Cdc6 and MCM occurs via the N-terminal portion of the MCM protein. It was found that Cdc6–MCM interaction, but not Cdc6–DNA binding, plays the predominant role in regulating MCM helicase activity. In addition, the data showed that the interactions with MCM modulate the autophosphorylation of Cdc6-1 and -2. The results also suggest that MCM and DNA may compete for Cdc6-1 protein binding. The implications of these observations for the initiation of archaeal DNA replication are discussed.     

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.     

Domain C of human poly(ADP-ribose) polymerase-1 is important for enzyme activity and contains a novel zinc-ribbon motif

Poly(ADP-ribose) polymerase-1 (PARP-1) is a multimodular nuclear protein that participates in many fundamental cellular activities. Stimulated by binding to nicked DNA, PARP-1 catalyzes poly(ADP-ribosyl)ation of the acceptor proteins using NAD (+) as a substrate. In this work, NMR methods were used to determine the solution structure of human PARP-1 protein. Domain C was found to contain a zinc-binding motif of three antiparallel beta-strands with four conserved cysteines positioned to coordinate the metal ligand, in addition to a helical region. The zinc-binding motif is structurally reminiscent of the "zinc-ribbon" fold, but with a novel spacing between the conserved cysteines (CX2CX12CX 9C). Domain C alone does not appear to bind to DNA. Interestingly, domain C is essential for PARP-1 activity, since a mixture containing nicked DNA and the PARP-1 ABDEF domains has only basal enzymatic activity, while the addition of domain C to the mixture initiated NAD (+) hydrolysis and the formation of poly(ADP-ribose), as detected by an NMR-based assay and autoradiography. The structural model for domain C in solution provides an important framework for further studies aimed at improving our understanding of how the various domains within the complex PARP-1 enzyme play their respective roles in regulating the enzyme activity when cells are under conditions of genotoxic stress.     

A CCCH zinc finger conserved in a replication protein a homolog found in diverse Euryarchaeotes

We describe a CCCH type of zinc finger domain in a replication protein A (RPA) homolog found in members of different lineages of the Euryarchaeota, a subdomain of Archaea. The zinc finger is characterized by CX(2)CX(8)CX(2)H, where X is any amino acid. Using MacRPA3, a representative of this new group of RPA in Methanosarcina acetivorans, we made two deletion mutants: a C-terminal deletion mutant lacking the zinc finger and an N-terminal deletion mutant containing the zinc finger domain. Whereas the N-terminal deletion mutant contained zinc at a level comparable to the wild-type protein level, the C-terminal deletion mutant was devoid of zinc. We further created four different mutants of MacRPA3 by replacing each of the four invariable amino acids in the zinc finger with alanine. Each single mutation at an invariable position resulted in a protein containing less than 35% of the zinc found in the wild-type protein. Circular dichroism spectra suggested that although the mutation at the first cysteine resulted in minor perturbation of protein structure, mutations at the other invariable positions led to larger structural changes. All proteins harboring a mutation at one of the invariable positions bound to single-stranded DNA weakly, and this translated into reduced capacity to stimulate DNA synthesis by M. acetivorans DNA polymerase BI. By subjecting the protein and its mutants to oxidizing and reducing conditions, we demonstrated that ssDNA binding by MacRPA3 may be regulated by redox through the zinc finger. Thus, the zinc finger modules in euryarchaeal RPA proteins may serve as a means by which the function of these proteins is regulated in the cell.     

Coupling of DNA binding and helicase activity is mediated by a conserved loop in the MCM protein

Minichromosome maintenance (MCM) helicases are the presumptive replicative helicases, thought to separate the two strands of chromosomal DNA during replication. In archaea, the catalytic activity resides within the C-terminal region of the MCM protein. In Methanothermobacter thermautotrophicus the N-terminal portion of the protein was shown to be involved in protein multimerization and binding to single and double stranded DNA. MCM homologues from many archaeal species have highly conserved predicted amino acid similarity in a loop located between β7 and β8 in the N-terminal part of the molecule. This high degree of conservation suggests a functional role for the loop. Mutational analysis and biochemical characterization of the conserved residues suggest that the loop participates in communication between the N-terminal portion of the helicase and the C-terminal catalytic domain. Since similar residues are also conserved in the eukaryotic MCM proteins, the data presented here suggest a similar coupling between the N-terminal and catalytic domain of the eukaryotic enzyme.     

An arginine residue instead of a conserved leucine residue in the recognition helix of the finger 3 of Zif268 stabilizes the domain structure and mediates DNA binding

The Cys(2)His(2)-type zinc finger is a common DNA binding motif that is widely used in the design of artificial zinc finger proteins. In almost all Cys(2)His(2)-type zinc fingers, position 4 of the α-helical DNA-recognition site is occupied by a Leu residue involved in formation of the minimal hydrophobic core. However, the third zinc finger domain of native Zif268 contains an Arg residue instead of the conserved Leu. Our aim in the present study was to clarify the role of this Arg in the formation of a stable domain structure and in DNA binding by substituting it with a Lys, Leu, or Hgn, which have different terminal side-chain structures. Assessed were the metal binding properties, peptide conformations, and DNA-binding abilities of the mutants. All three mutant finger 3 peptides exhibited conformations and thermal stabilities similar to the wild-type peptide. In DNA-binding assays, the Lys mutant bound to target DNA, though its affinity was lower than that of the wild-type peptide. On the other hand, the Leu and Hgn mutants had no ability to bind DNA, despite the similarity in their secondary structures to the wild-type. Our results demonstrate that, as with the Leu residue, the aliphatic carbon side chain of this Arg residue plays a key role in the formation of a stable zinc finger domain, and its terminal guanidinium group appears to be essential for DNA binding mediated through both electrostatic interaction and hydrogen bonding with DNA phosphate backbone.     

Replication factors MCM2 and ORC1 interact with the histone acetyltransferase HBO1

The minichromosome maintenance (MCM) proteins, together with the origin recognition complex (ORC) proteins and Cdc6, play an essential role in eukaryotic DNA replication through the formation of a pre-replication complex at origins of replication. We used a yeast two-hybrid screen to identify MCM2-interacting proteins. One of the proteins we identified is identical to the ORC1-interacting protein termed HBO1. HBO1 belongs to the MYST family, characterized by a highly conserved C2HC zinc finger and a putative histone acetyltransferase domain. Biochemical studies confirmed the interaction between MCM2 and HBO1 in vitro and in vivo. An N-terminal domain of MCM2 is necessary for binding to HBO1, and a C2HC zinc finger of HBO1 is essential for binding to MCM2. A reverse yeast two-hybrid selection was performed to isolate an allele of MCM2 that is defective for interaction with HBO1; this allele was then used to isolate a suppressor mutant of HBO1 that restores the interaction with the mutant MCM2. This suppressor mutation was located in the HBO1 zinc finger. Taken together, these findings strongly suggest that the interaction between MCM2 and HBO1 is direct and mediated by the C2HC zinc finger of HBO1. The biochemical and genetic interactions of MYST family protein HBO1 with two components of the replication apparatus, MCM2 and ORC1, suggest that HBO1-associated HAT activity may play a direct role in the process of DNA replication.     

The zinc-binding motif in tankyrases is required for the structural integrity of the catalytic ADP-ribosyltransferase domain

Tankyrases are ADP-ribosylating enzymes that regulate many physiological processes in the cell and are considered promising drug targets for cancer and fibrotic diseases. The catalytic ADP-ribosyltransferase domain of tankyrases contains a unique zinc-binding motif of unknown function. Recently, this motif was suggested to be involved in the catalytic activity of tankyrases. In this work, we set out to study the effect of the zinc-binding motif on the activity, stability and structure of human tankyrases. We generated mutants of human tankyrase (TNKS) 1 and TNKS2, abolishing the zinc-binding capabilities, and characterized the proteins biochemically and biophysically in vitro. We further generated a crystal structure of TNKS2, in which the zinc ion was oxidatively removed. Our work shows that the zinc-binding motif in tankyrases is a crucial structural element which is particularly important for the structural integrity of the acceptor site. While mutation of the motif rendered TNKS1 inactive, probably due to introduction of major structural defects, the TNKS2 mutant remained active and displayed an altered activity profile compared to the wild-type.     

The NMR solution structure of the 30S ribosomal protein S27e encoded in gene RS27_ARCFU of Archaeoglobus fulgidis reveals a novel protein fold

The Archaeoglobus fulgidis gene RS27_ARCFU encodes the 30S ribosomal protein S27e. Here, we present the high-quality NMR solution structure of this archaeal protein, which comprises a C4 zinc finger motif of the CX(2)CX(14-16)CX(2)C class. S27e was selected as a target of the Northeast Structural Genomics Consortium (target ID: GR2), and its three-dimensional structure is the first representative of a family of more than 116 homologous proteins occurring in eukaryotic and archaeal cells. As a salient feature of its molecular architecture, S27e exhibits a beta-sandwich consisting of two three-stranded sheets with topology B(decreasing), A(increasing), F(decreasing), and C(increasing), D(decreasing), E(increasing). Due to the uniqueness of the arrangement of the strands, the resulting fold was found to be novel. Residues that are highly conserved among the S27 proteins allowed identification of a structural motif of putative functional importance; a conserved hydrophobic patch may well play a pivotal role for functioning of S27 proteins, be it in archaeal or eukaryotic cells. The structure of human S27, which possesses a 26-residue amino-terminal extension when compared with the archaeal S27e, was modeled on the basis of two structural templates, S27e for the carboxy-terminal core and the amino-terminal segment of the archaeal ribosomal protein L37Ae for the extension. Remarkably, the electrostatic surface properties of archaeal and human proteins are predicted to be entirely different, pointing at either functional variations among archaeal and eukaryotic S27 proteins, or, assuming that the function remained invariant, to a concerted evolutionary change of the surface potential of proteins interacting with S27.     

The zinc finger motif of Escherichia coli RecQ is implicated in both DNA binding and protein folding

The RecQ family of DNA helicases has been shown to be important for the maintenance of genomic integrity. Mutations in human RecQ genes lead to genomic instability and cancer. Several RecQ family of helicases contain a putative zinc finger motif of the C4 type at the C terminus that has been identified in the crystalline structure of RecQ helicase from Escherichia coli. To better understand the role of this motif in helicase from E. coli, we constructed a series of single mutations altering the conserved cysteines as well as other highly conserved residues. All of the resulting mutant proteins exhibited a high level of susceptibility to degradation, making functional analysis impossible. In contrast, a double mutant protein in which both cysteine residues Cys397 and Cys400 in the zinc finger motif were replaced by asparagine residues was purified to homogeneity. Slight local conformational changes were detected, but the rest of the mutant protein has a well defined tertiary structure. Furthermore, the mutant enzyme displayed ATP binding affinity similar to the wild-type enzyme but was severely impaired in DNA binding and in subsequent ATPase and helicase activities. These results revealed that the zinc finger binding motif is involved in maintaining the integrity of the whole protein as well as DNA binding. We also showed that the zinc atom is not essential to enzymatic activity.