Snapshots of a Y-family DNA polymerase in replication: substrate-induced conformational transitions and implications for fidelity of Dpo4

Y-family DNA polymerases catalyze translesion DNA synthesis over damaged DNA. Each Y-family polymerase has a polymerase core consisting of a palm, finger and thumb domain in addition to a fourth domain known as a little finger domain. It is unclear how each domain moves during nucleotide incorporation and what type of conformational changes corresponds to the rate-limiting step previously reported in kinetic studies. Here, we present three crystal structures of the prototype Y-family polymerase: apo-Dpo4 at 1.9 Å resolution, Dpo4-DNA binary complex and Dpo4-DNA-dTMP ternary complex at 2.2 Å resolution. Dpo4 undergoes dramatic conformational changes from the apo to the binary structures with a 131° rotation of the little finger domain relative to the polymerase core upon DNA binding. This DNA-induced conformational change is verified in solution by our tryptophan fluorescence studies. In contrast, the polymerase core retains the same conformation in all three conformationally distinct states. Particularly, the finger domain which is responsible for checking base pairing between the template base and an incoming nucleotide retains a rigid conformation. The inflexibility of the polymerase core likely contributes to the low fidelity of Dpo4, in addition to its loose and solvent-accessible active site. Interestingly, while the binary and ternary complexes of Dpo4 retain an identical global conformation, the aromatic side chains of two conserved tyrosines at the nucleotide-binding site change orientations between the binary and ternary structures. Such local conformational changes may correspond to the rate-limiting step in the mechanism of nucleotide incorporation. Together, the global and local conformational transitions observed in our study provide a structural basis for the distinct kinetic steps of a catalytic cycle of DNA polymerization performed by a Y-family polymerase.     

Synthesis and crystal structures of cobalt(II), cadmium(II), and zinc(II) complexes of 4-nitro phenylcyanamide: enhancing the biological properties through bound to human serum albumin

Metal complexes of the type [Co(phen)₂(4-NO₂pcyd)₂].CH₃OH, 1, [Zn(phen)₂(4-NO₂pcyd)₂].CH₃OH, 2, [Cd(phen)₂(4-NO₂pcyd)₂], and 3, (phen?=?1,10-phenanthroline, 4-NO₂pcyd?=?4-nitro phenylcyanamide) have been studied. The synthesis, characterization, and the biological activities of complexes 1-3 have been investigated. The geometries of complexes 1-3 were confirmed by single-crystal X-ray crystallography. The interactions of complexes 1-3 with human serum albumin (HSA) were studied using fluorescence and circular dichroism spectroscopy. The thermodynamic studies have showed the reaction for the binding of complexes 1-3 with HSA is hydrophobic (ΔH_0???0 and ΔS₀ > 0). The in vitro cytotoxic potential of complexes 1-3 and their complexes with HSA were examined. The complexes 1-3 with HSA enhance about 3-fold cytotoxicity in cancer cells lines.     

The Interaction between Polynucleotide Kinase Phosphatase and the DNA Repair Protein XRCC1 Is Critical for Repair of DNA Alkylation Damage and Stable Association at DNA Damage Sites

XRCC1 plays a key role in the repair of DNA base damage and single-strand breaks. Although it has no known enzymatic activity, XRCC1 interacts with multiple DNA repair proteins and is a subunit of distinct DNA repair protein complexes. Here we used the yeast two-hybrid genetic assay to identify mutant versions of XRCC1 that are selectively defective in interacting with a single protein partner. One XRCC1 mutant, A482T, that was defective in binding to polynucleotide kinase phosphatase (PNKP) not only retained the ability to interact with partner proteins that bind to different regions of XRCC1 but also with aprataxin and aprataxin-like factor whose binding sites overlap with that of PNKP. Disruption of the interaction between PNKP and XRCC1 did not impact their initial recruitment to localized DNA damage sites but dramatically reduced their retention there. Furthermore, the interaction between PNKP and the DNA ligase IIIα-XRCC1 complex significantly increased the efficiency of reconstituted repair reactions and was required for complementation of the DNA damage sensitivity to DNA alkylation agents of xrcc1 mutant cells. Together our results reveal novel roles for the interaction between PNKP and XRCC1 in the retention of XRCC1 at DNA damage sites and in DNA alkylation damage repair.     

Termination of DNA replication at Tus-ter barriers results in under-replication of template DNA

The complete and accurate duplication of genomic information is vital to maintain genome stability in all domains of life. In Escherichia coli, replication termination, the final stage of the duplication process, is confined to the “replication fork trap” region by multiple unidirectional fork barriers formed by the binding of Tus protein to genomic ter sites. Termination typically occurs away from Tus-ter complexes, but they become part of the fork fusion process when a delay to one replisome allows the second replisome to travel more than halfway around the chromosome. In this instance, replisome progression is blocked at the nonpermissive interface of the Tus-ter complex, termination then occurs when a converging replisome meets the permissive interface. To investigate the consequences of replication fork fusion at Tus-ter complexes, we established a plasmid-based replication system where we could mimic the termination process at Tus-ter complexes in vitro. We developed a termination mapping assay to measure leading strand replication fork progression and demonstrate that the DNA template is under-replicated by 15 to 24 bases when replication forks fuse at Tus-ter complexes. This gap could not be closed by the addition of lagging strand processing enzymes or by the inclusion of several helicases that promote DNA replication. Our results indicate that accurate fork fusion at Tus-ter barriers requires further enzymatic processing, highlighting large gaps that still exist in our understanding of the final stages of chromosome duplication and the evolutionary advantage of having a replication fork trap.     

Modulation of T4 gene 32 protein DNA binding activity by the recombination mediator protein UvsY

Bacteriophage T4 UvsY is a recombination mediator protein that promotes assembly of the UvsX-ssDNA presynaptic filament. UvsY helps UvsX to displace T4 gene 32 protein (gp32) from ssDNA, a reaction necessary for proper formation of the presynaptic filament. Here we use DNA stretching to examine UvsY interactions with single DNA molecules in the presence and absence of gp32 and a gp32 C-terminal truncation (*I), and show that in both cases UvsY is able to destabilize gp32-ssDNA interactions. In these experiments UvsY binds more strongly to dsDNA than ssDNA due to its inability to wrap ssDNA at high forces. To support this hypothesis, we show that ssDNA created by exposure of stretched DNA to glyoxal is strongly wrapped by UvsY, but wrapping occurs only at low forces. Our results demonstrate that UvsY interacts strongly with stretched DNA in the absence of other proteins. In the presence of gp32 and *I, UvsY is capable of strongly destabilizing gp32-DNA complexes in order to facilitate ssDNA wrapping, which in turn prepares the ssDNA for presynaptic filament assembly in the presence of UvsX. Thus, UvsY mediates UvsX binding to ssDNA by converting rigid gp32-DNA filaments into a structure that can be strongly bound by UvsX.     

Carcinoembryonic antigen promotes colorectal cancer progression by targeting adherens junction complexes

Oncomarkers play important roles in the detection and management of human malignancies. Carcinoembryonic antigen (CEA, CEACAM5) and epithelial cadherin (E-cadherin) are considered as independent tumor markers in monitoring metastatic colorectal cancer. They are both expressed by cancer cells and can be detected in the blood serum. We investigated the effect of CEA production by MIP101 colorectal carcinoma cell lines on E-cadherin adherens junction (AJ) protein complexes. No direct interaction between E-cadherin and CEA was detected; however, the functional relationships between E-cadherin and its AJ partners: α-, β- and p120 catenins were impaired. We discovered a novel interaction between CEA and beta-catenin protein in the CEA producing cells. It is shown in the current study that CEA overexpression alters the splicing of p120 catenin and triggers the release of soluble E-cadherin. The influence of CEA production by colorectal cancer cells on the function of E-cadherin junction complexes may explain the link between the elevated levels of CEA and the increase in soluble E-cadherin during the progression of colorectal cancer.     

In vitro assembly of semi-artificial molecular machine and its use for detection of DNA damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc). This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage. Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks), fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO(4) blunt-ended break (DNase I-type breaks), if T4 DNA ligase is present in the solution. When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO(4) blunt-ended breaks, the ligase reacts with 5'PO(4) DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO(4) blunt-ended DNA breaks. This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.     

Dynamic Regulatory Interactions of Rad51, Rad52, and Replication Protein-A in Recombination Intermediates

Rad51, Rad52, and replication protein-A (RPA) play crucial roles in the repair of DNA double-strand breaks in Saccharomyces cerevisiae. Rad51 mediates DNA strand exchange, a key reaction in DNA recombination. Rad52 recruits Rad51 into single-stranded DNAs (ssDNAs) that are saturated with RPA. Rad52 also promotes annealing of ssDNA strands that are complexed with RPA. Specific protein-protein interactions are involved in these reactions. Here we report new biochemical characteristics of these protein interactions. First, Rad52-RPA interaction requires multiple molecules of RPA to be associated with ssDNA, suggesting that multiple contacts between the Rad52 ring and RPA-ssDNA filament are needed for stable binding. Second, RPA-t11, which is a recombination-deficient mutant of RPA, displays a defect in interacting with Rad52 in the presence of salt above 50 mM, explaining the defect in Rad52-mediated ssDNA annealing in the presence of this mutation. Third, ssDNA annealing promoted by Rad52 is preceded by aggregation of multiple RPA-ssDNA complexes with Rad52, and Rad51 inhibits this aggregation. These results suggest a regulatory role for Rad51 that suppresses ssDNA annealing and facilitates DNA strand invasion. Finally, the Rad51-double-stranded DNA complex disrupts Rad52-RPA interaction in ssDNA and titrates Rad52 from RPA. This suggests an additional regulatory role for Rad51 following DNA strand invasion, where Rad51-double-stranded DNA may inhibit illegitimate second-end capture to ensure the error-free repair of a DNA double-strand break.     

Ascorbate Efflux as a New Strategy for Iron Reduction and Transport in Plants

Iron (Fe) is essential for virtually all living organisms. The identification of the chemical forms of iron (the speciation) circulating in and between cells is crucial to further understand the mechanisms of iron delivery to its final targets. Here we analyzed how iron is transported to the seeds by the chemical identification of iron complexes that are delivered to embryos, followed by the biochemical characterization of the transport of these complexes by the embryo, using the pea (Pisum sativum) as a model species. We have found that iron circulates as ferric complexes with citrate and malate (Fe(III)₃Cit₂Mal₂, Fe(III)₃Cit₃Mal₁, Fe(III)Cit₂). Because dicotyledonous plants only transport ferrous iron, we checked whether embryos were capable of reducing iron of these complexes. Indeed, embryos did express a constitutively high ferric reduction activity. Surprisingly, iron(III) reduction is not catalyzed by the expected membrane-bound ferric reductase. Instead, embryos efflux high amounts of ascorbate that chemically reduce iron(III) from citrate-malate complexes. In vitro transport experiments on isolated embryos using radiolabeled ⁵⁵Fe demonstrated that this ascorbate-mediated reduction is an obligatory step for the uptake of iron(II). Moreover, the ascorbate efflux activity was also measured in Arabidopsis embryos, suggesting that this new iron transport system may be generic to dicotyledonous plants. Finally, in embryos of the ascorbate-deficient mutants vtc2-4, vtc5-1, and vtc5-2, the reducing activity and the iron concentration were reduced significantly. Taken together, our results identified a new iron transport mechanism in plants that could play a major role to control iron loading in seeds.     

Seven new species within western Atlantic Starksia atlantica, S. lepicoelia, and S. sluiteri (Teleostei, Labrisomidae), with comments on congruence of DNA barcodes and species

Specimens of Starksia were collected throughout the western Atlantic, and a 650-bp portion of the mitochondrial gene cytochrome oxidase-c subunit I (COl) was sequenced as part of a re-analysis of species diversity of western Central Atlantic shorefishes. A neighbor-joining tree constructed from the sequence data suggests the existence of several cryptic species. Voucher specimens from each genetically distinct lineage and color photographs of vouchers taken prior to dissection and preservation were examined for diagnostic morphological characters. The results suggest that Starksia atlantica, Starksia lepicoelia, and Starksia sluiteri are species complexes, and each comprises three or more species. Seven new species are described. DNA data usually support morphological features, but some incongruence between genetic and morphological data exists. Genetic lineages are only recognized as species if supported by morphology. Genetic lineages within western Atlantic Starksia generally correspond to geography, such that members of each species complex have a very restricted geographical distribution. Increasing geographical coverage of sampling locations will almost certainly increase the number of Starksia species and species complexes recognized in the western Atlantic. Combining molecular and morphological investigations is bringing clarity to the taxonomy of many genera of morphologically similar fishes and increasing the number of currently recognized species. Future phylogenetic studies should help resolve species relationships and shed light on patterns of speciation in western Atlantic Starksia.     

Conformational Adaptability of Redβ during DNA Annealing and Implications for Its Structural Relationship with Rad52

Single-strand annealing proteins, such as Redβ from λ phage or eukaryotic Rad52, play roles in homologous recombination. Here, we use atomic force microscopy to examine Redβ quaternary structure and Redβ-DNA complexes. In the absence of DNA, Redβ forms a shallow right-handed helix. The presence of single-stranded DNA (ssDNA) disrupts this structure. Upon addition of a second complementary ssDNA, annealing generates a left-handed helix that incorporates 14 Redβ monomers per helical turn, with each Redβ monomer annealing ≈ 11 bp of DNA. The smallest stable annealing intermediate requires 20 bp DNA and two Redβ monomers. Hence, we propose that Redβ promotes base pairing by first increasing the number of transient interactions between ssDNAs. Then, annealing is promoted by the binding of a second Redβ monomer, which nucleates the formation of a stable annealing intermediate. Using threading, we identify sequence similarities between the RecT/Redβ and the Rad52 families, which strengthens previous suggestions, based on similarities of their quaternary structures, that they share a common mode of action. Hence, our findings have implications for a common mechanism of DNA annealing mediated by single-strand annealing proteins including Rad52.     

Different Behaviors In Vivo of Mutations in the β Hairpin Loop of the DNA Polymerases of the Closely Related Phages T4 and RB69

The T4 and RB69 DNA replicative polymerases are members of the B family and are highly similar. Both replicate DNA with high fidelity and employ the same mechanism that allows efficient switching of the primer terminus between the polymerase and exonuclease sites. Both polymerases have a β hairpin loop (hereafter called the β loop) in their exonuclease domains that plays an important role in active-site switching. The β loop is involved in strand separation and is needed to stabilize partially strand-separated exonuclease complexes. In T4 DNA polymerase, modification of the β-loop residue G255 to Ser confers a strong mutator phenotype in vivo due to a reduced ability to form editing complexes. Here, we describe the RB69 DNA polymerase mutant with the equivalent residue (G258) changed to Ser but showing only mild mutator activity in vivo. On the other hand, deletion of the tip of the RB69 β loop confers a strong mutator phenotype in vivo. Based on detailed mutational spectral analyses, DNA binding activities, and coupled polymerase/exonuclease assays, we define the differences between the T4 and RB69 polymerases. We propose that their β loops facilitate strand separation in both polymerases, while the residues that form the loop have low structural constraints.     

APLF promotes the assembly and activity of non‐homologous end joining protein complexes

Non‐homologous end joining (NHEJ) is critical for the maintenance of genetic integrity and DNA double‐strand break (DSB) repair. NHEJ is regulated by a series of interactions between core components of the pathway, including Ku heterodimer, XLF/Cernunnos, and XRCC4/DNA Ligase 4 (Lig4). However, the mechanisms by which these proteins assemble into functional protein–DNA complexes are not fully understood. Here, we show that the von Willebrand (vWA) domain of Ku80 fulfills a critical role in this process by recruiting Aprataxin‐and‐PNK‐Like Factor (APLF) into Ku‐DNA complexes. APLF, in turn, functions as a scaffold protein and promotes the recruitment and/or retention of XRCC4‐Lig4 and XLF, thereby assembling multi‐protein Ku complexes capable of efficient DNA ligation in vitro and in cells. Disruption of the interactions between APLF and either Ku80 or XRCC4‐Lig4 disrupts the assembly and activity of Ku complexes, and confers cellular hypersensitivity and reduced rates of chromosomal DSB repair in avian and human cells, respectively. Collectively, these data identify a role for the vWA domain of Ku80 and a molecular mechanism by which DNA ligase proficient complexes are assembled during NHEJ in mammalian cells, and reveal APLF to be a structural component of this critical DSB repair pathway.     

On the interactions of histone H4 and H4 peptides with DNA. Electrooptical, hydrodynamic and electron microscopy studies

The interactions of DNA with histone H4 and with its fragments N-H4 (1-84) and C-H4 (85-102) have been studied by using electrooptical techniques, viscosity and electron microscopy. Electron microscopy reveals that histone H4 induces a large folding of DNA molecules : this is in agreement with electrooptical measurements which indicate that, with the increase of their ratio, H4/DNA complexes undergo a gradual process of condensation. Viscosity measurements show that complexes at ratios up to 0.20-0.25 become more rigid as compared to DNA. It appears that C-H4, and not the N-H4 fragment, causes a great distorsion to the structure of DNA, accompanied by an increase of rigidity at ratios up to 0.20-0.25, as occurs for H4/DNA complexes. Electrooptical studies of C-H4/DNA complexes show, along a range of histone/DNA ratios, an important permanent dipole component. These effects reveal a particular mode of interaction of C-H4 with DNA, indicating that some charged residues of the peptide are kept distant enough from the DNA backbone. As no dipole character, in addition to that shown for DNA, has been detected for H4/DNA complexes, it is concluded that the conformation of the H4 molecule modifies to some extent the interaction of the C-terminal region. Our results show that this histone, and particularly its C-terminal region, is important as a determinant factor in the folding of DNA within artificial complexes.     

The Werner syndrome exonuclease facilitates DNA degradation and high fidelity DNA polymerization by human DNA polymerase δ

DNA Polymerase δ (Pol δ) and the Werner syndrome protein, WRN, are involved in maintaining cellular genomic stability. Pol δ synthesizes the lagging strand during replication of genomic DNA and also functions in the synthesis steps of DNA repair and recombination. WRN is a member of the RecQ helicase family, loss of which results in the premature aging and cancer-prone disorder, Werner syndrome. Both Pol δ and WRN encode 3' → 5' DNA exonuclease activities. Pol δ exonuclease removes 3'-terminal mismatched nucleotides incorporated during replication to ensure high fidelity DNA synthesis. WRN exonuclease degrades DNA containing alternate secondary structures to prevent formation and enable resolution of stalled replication forks. We now observe that similarly to WRN, Pol δ degrades alternate DNA structures including bubbles, four-way junctions, and D-loops. Moreover, WRN and Pol δ form a complex with enhanced ability to hydrolyze these structures. We also present evidence that WRN can proofread for Pol δ; WRN excises 3'-terminal mismatches to enable primer extension by Pol δ. Consistent with our in vitro observations, we show that WRN contributes to the maintenance of DNA synthesis fidelity in vivo. Cells expressing limiting amounts (～10% of normal) of WRN have elevated mutation frequencies compared with wild-type cells. Together, our data highlight the importance of WRN exonuclease activity and its cooperativity with Pol δ in preserving genome stability, which is compromised by the loss of WRN in Werner syndrome.     

DNA topology in a chromatin model system

The synthetic copolypeptide (Lys³³, Leu⁶⁷)₁₀₀-Orn₂₀, modeled on some general features of the histone sequences, has been found to supercoil the DNA double helix, wrapping it into a micelle, as a result of cohesive interactions between the polypeptide hydrophobic moieties. X-ray low-angle diffraction of complexes between the polypeptide and DNA is characterized by maxima at 50, 32, and 23 Å, reminiscent of the chromatin pattern. The existence of a nucleosome-like structure along the DNA is suggested by gel electrophoresis analysis of DNA fragments after micrococcal nuclease digestion, showing the presence of a fragment of about 100 basepairs (bp) long. Topological experiments on the complexes with supercoiled as well as relaxed circular DNA by two-dimentional gel electrophoresis show the presence of left-handed superhelical turns. The results are in agreement with an intrinsic propensity of B-DNA to writhe into left-handed supercoils.     

Response of the Bacteriophage T4 Replisome to Noncoding Lesions and Regression of a Stalled Replication Fork

DNA is constantly damaged by endogenous and exogenous agents. The resulting DNA lesions have the potential to halt the progression of the replisome, possibly leading to replication fork collapse. Here, we examine the effect of a noncoding DNA lesion in either leading strand template or lagging strand template on the bacteriophage T4 replisome. A damaged base in the lagging strand template does not affect the progression of the replication fork. Instead, the stalled lagging strand polymerase recycles from the lesion and initiates the synthesis of a new Okazaki fragment upstream of the damaged base. In contrast, when the replisome encounters a blocking lesion in the leading strand template, the replication fork only travels approximately 1 kb beyond the point of the DNA lesion before complete replication fork collapse. The primosome and the lagging strand polymerase remain active during this period, and an Okazaki fragment is synthesized beyond the point of the leading strand lesion. There is no evidence for a new priming event on the leading strand template. Instead, the DNA structure that is produced by the stalled replication fork is a substrate for the DNA repair helicase UvsW. UvsW catalyzes the regression of a stalled replication fork into a “chicken-foot” structure that has been postulated to be an intermediate in an error-free lesion bypass pathway.     

Synthesis, characterization, thermal behavior, and DNA-cleaving studies of cyano-bridged nickel(II)-copper(II) complexes of 4-(pyridin-2-ylazenyl)resorcinol

We present here the syntheses of a mononuclear Cu^II complex and two polynuclear Cu^II Ni^II complexes of the azenyl ligand, 4‐(pyridin‐2‐ylazenyl)resorcinol (HL; 1). The reaction of HL ( 1 ) and copper(II) perchlorate with KCN gave a mononuclear complex [CuL(CN)] ( 4 ). Using 4 , one pentanuclear complex, [{CuL(NC)}₄Ni](ClO₄)₂ ( 5 ) and one trinuclear complex, [{CuL(CN)}₂NiL]ClO₄ ( 6 ), were prepared and characterized by elemental analyses, magnetic susceptibility, molar conductance, IR, and thermal analysis. Stoichiometric and spectral results of the mononuclear Cu^II complex indicated that the metal/ligand/CN ratio was 1 : 1 : 1, and the ligand behaved as a tridentate ligand forming neutral metal chelates through the pyridinyl and azenyl N‐, and resorcinol O‐atom. The interaction between the compounds (the ligand 1 , its Ni^II and Cu^II complexes without CN, i.e., 2 and 3 , and its complexes with CN, 4 – 6 ) and DNA has also been investigated by agarose gel electrophoresis. The pentanuclear Cu₄Ni complex ( 5 ) with H₂O₂ as a co‐oxidant exhibited the strongest DNA‐cleaving activity.     

Two crystal forms of the restriction enzyme MspI-DNA complex show the same novel structure

The crystal structure of the Type IIP restriction endonuclease MspI bound to DNA containing its cognate recognition sequence has been determined in both monoclinic and orthorhombic space groups. Significantly, these two independent crystal forms present an identical structure of a novel monomer-DNA complex, suggesting a functional role for this novel enzyme-DNA complex. In both crystals, MspI interacts with the CCGG DNA recognition sequence as a monomer, using an asymmetric mode of recognition by two different structural motifs in a single polypeptide. In the crystallographic asymmetric unit, the two DNA molecules in the two MspI-DNA complexes appear to stack with each other forming an end-to-end pseudo-continuous 19-mer duplex. They are primarily B-form and no major bends or kinks are observed. For DNA recognition, most of the specific contacts between the enzyme and the DNA are preserved in the orthorhombic structure compared with the monoclinic structure. A cation is observed near the catalytic center in the monoclinic structure at a position homologous to the 74/45 metal site of EcoRV, and the orthorhombic structure also shows signs of this same cation. However, the coordination ligands of the metal are somewhat different from those of the 74/45 metal site of EcoRV. Combined with structural information from other solved structures of Type II restriction enzymes, the possible relationship between the structures of the enzymes and their cleavage behaviors is discussed.     

Direct observation of translocation in individual DNA polymerase complexes

Complexes of phi29 DNA polymerase and DNA fluctuate on the millisecond time scale between two ionic current amplitude states when captured atop the α-hemolysin nanopore in an applied field. The lower amplitude state is stabilized by complementary dNTP and thus corresponds to complexes in the post-translocation state. We have demonstrated that in the upper amplitude state, the DNA is displaced by a distance of one nucleotide from the post-translocation state. We propose that the upper amplitude state corresponds to complexes in the pre-translocation state. Force exerted on the template strand biases the complexes toward the pre-translocation state. Based on the results of voltage and dNTP titrations, we concluded through mathematical modeling that complementary dNTP binds only to the post-translocation state, and we estimated the binding affinity. The equilibrium between the two states is influenced by active site-proximal DNA sequences. Consistent with the assignment of the upper amplitude state as the pre-translocation state, a DNA substrate that favors the pre-translocation state in complexes on the nanopore is a superior substrate in bulk phase for pyrophosphorolysis. There is also a correlation between DNA sequences that bias complexes toward the pre-translocation state and the rate of exonucleolysis in bulk phase, suggesting that during DNA synthesis the pathway for transfer of the primer strand from the polymerase to exonuclease active site initiates in the pre-translocation state.