Structure of an open conformation of T7 DNA Polymerase reveals novel structural features regulating primer-template stabilization at the polymerization active-site

The crystal structure of full-length T7 DNA polymerase in complex with its processivity factor thioredoxin and double-stranded DNA in the polymerization active site exhibits two novel structural motifs in family-A DNA polymerases: an extended b-hairpin at the fingers subdomain, that interacts with the DNA template strand downstream the primer-terminus, and a helix-loop-helix motif (insertion1) located between residues 102 to 122 in the exonuclease domain. The extended b-hairpin is involved in nucleotide incorporation on substrates with 5'-overhangs longer than 2 nucleotides, suggesting a role in stabilizing the template strand into the polymerization domain. Our biochemical data reveal that insertion1 of the exonuclease domain makes stabilizing interactions that facilitate proofreading by shuttling the primer strand into the exonuclease active site. Overall, our studies evidence conservation of the 3'-5' exonuclease domain fold between family-A DNA polymerases and highlight the modular architecture of T7 DNA polymerase. Our data suggest that the intercalating b-hairpin guides the template-strand into the polymerization active site after the T7 primase-helicase unwinds the DNA double helix ameliorating the formation of secondary structures and decreasing the appearance of indels

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Accuracy, lesion bypass, strand displacement and translocation by DNA polymerases

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2003.1374 ◽

2004 ◽

Vol 359 (1441) ◽

pp. 17-23 ◽

Cited By ~ 33

Author(s):

Thomas A. Steitz ◽

Y. Whitney Yin

Keyword(s):

Conformational Change ◽

Dna Polymerase ◽

Active Site ◽

Dna Polymerases ◽

Lesion Bypass ◽

Dna Structures ◽

Template Strand ◽

Pol I ◽

Deoxynucleoside Triphosphate ◽

Copy Dna

The structures of DNA polymerases from different families show common features and significant differences that shed light on the ability of these enzymes to accurately copy DNA and translocate. The structure of a B family DNA polymerase from phage RB69 exhibits an active–site closing conformational change in the fingers domain upon forming a ternary complex with primer template in deoxynucleoside triphosphate. The rotation of the fingers domain α–helices by 60° upon dNTP binding is analogous to the changes seen in other families of polymerases. When the 3' terminus is bound to the editing 3' exonuclease active site, the orientation of the DNA helix axis changes by 40° and the thumb domain re–orients with the DNA. Structures of substrate and product complexes of T7 RNA polymerase, a structural homologue of T7 DNA polymerase, show that family polymerases use the rotation conformational change of the fingers domain to translocate down the DNA. The fingers opening rotation that results in translocation is powered by the release of the product pyrophosphate and also enables the Pol I family polymerases to function as a helicase in displacing the downstream non–template strand from the template strand.

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The Loop of the TPR1 Subdomain of Phi29 DNA Polymerase Plays a Pivotal Role in Primer-Terminus Stabilization at the Polymerization Active Site

Biomolecules ◽

10.3390/biom9110648 ◽

2019 ◽

Vol 9 (11) ◽

pp. 648

Author(s):

del Prado ◽

Santos ◽

Lázaro ◽

Salas ◽

de Vega

Keyword(s):

Dna Polymerase ◽

Active Site ◽

Structural Changes ◽

Binding Capacity ◽

Dna Polymerases ◽

Crystallographic Structure ◽

Genome Replication ◽

Terminal Protein ◽

Phi29 Dna Polymerase

Bacteriophage Phi29 DNA polymerase belongs to the protein-primed subgroup of family B DNA polymerases that use a terminal protein (TP) as a primer to initiate genome replication. The resolution of the crystallographic structure showed that it consists of an N-terminal domain with the exonuclease activity and a C-terminal polymerization domain. It also has two subdomains specific of the protein-primed DNA polymerases; the TP Regions 1 (TPR1) that interacts with TP and DNA, and 2 (TPR2), that couples both processivity and strand displacement to the enzyme. The superimposition of the structures of the apo polymerase and the polymerase in the polymerase/TP heterodimer shows that the structural changes are restricted almost to the TPR1 loop (residues 304–314). In order to study the role of this loop in binding the DNA and the TP, we changed the residues Arg306, Arg308, Phe309, Tyr310, and Lys311 into alanine, and also made the deletion mutant Δ6 lacking residues Arg306–Lys311. The results show a defective TP binding capacity in mutants R306A, F309A, Y310A, and Δ6. The additional impaired primer-terminus stabilization at the polymerization active site in mutants Y310A and Δ6 allows us to propose a role for the Phi29 DNA polymerase TPR1 loop in the proper positioning of the DNA and TP-priming 3’-OH termini at the preinsertion site of the polymerase to enable efficient initiation and further elongation steps during Phi29 TP-DNA replication.

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Water–DNA interactions as studied by X–ray and neutron fibre diffraction

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2004.1501 ◽

2004 ◽

Vol 359 (1448) ◽

pp. 1237-1248 ◽

Cited By ~ 47

Author(s):

Watson Fuller ◽

Trevor Forsyth ◽

Arumugam Mahendrasingam

Keyword(s):

Structural Information ◽

Double Helix ◽

Water Environment ◽

Structural Features ◽

Molecular Conformations ◽

X Ray ◽

Biochemical Processes ◽

Fibre Diffraction ◽

Dna Double Helix ◽

High Degree

X–ray fibre–diffraction studies indicate a high degree of stereochemical specificity in interactions between water and the DNA double helix. Evidence for this comes from data that show that the molecular conformations assumed by DNA in fibres are highly reproducible and that the hydration–driven transitions between these conformations are fully reversible. These conformational transitions are induced by varying the relative humidity of the fibre environment and hence its water content. Further evidence for stereochemical specificity comes from the observed dependence of the conformation assumed on the ionic content of the fibre and the nucleotide sequence of the DNA. For some transitions, information on stereochemical pathways has come from real–time X–ray fibre diffraction using synchrotron radiation; information on the location of water with respect to the double helix for a number of DNA conformations has come from neutron fibre diffraction. This structural information from fibre–diffraction studies of DNA is complemented by information from X–ray single–crystal studies of oligonucleotides. If the biochemical processes involving DNA have evolved to exploit the structural features observed in DNA fibres and oligonucleotide single crystals, the challenges in developing alternatives to a water environment can be expected to be very severe.

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Polyamine derivatives as potential bisintercalators with antiproliferative activity

Postępy Polskiej Medycyny i Farmacji ◽

10.5604/01.3001.0011.6389 ◽

2016 ◽

Vol 4 ◽

pp. 9-15

Author(s):

Marta Szumilak

Keyword(s):

Ethidium Bromide ◽

Antiproliferative Activity ◽

Double Helix ◽

Structural Features ◽

Discovery Studio ◽

Polycyclic Aromatic ◽

Ic50 Values ◽

Dna Double Helix ◽

Polyamine Derivatives

Bisntercalators are very interesting group of compounds with potential antitumor activity. They interact reversibly with DNA double helix. These agents share common structural features such as the presence of two, planar, polycyclic aromatic or heteroaromatic systems separated by a spacer chain which must be long enough to enable double intercalation between base pairs. The unique chemical structure of these compounds provides numerous modifications within their structure resulting either in higher activity or increased selectivity toward tumor cells. Within the framework of the project, new polyamine derivatives containing dimeric phthalimide, quinoline, cinnoline and chromone moieties were obtained. Three different polyamines: 1,4-bis(3-aminopropyl)piperazine, 4,9-dioxa-1,12-dodecanediamine, 3,3’-diamino-N-methyldipropylamine were used as linkers. The biological activity of compounds was assessed in vitro in a highly aggressive melanoma cell line A375. Quinoline derivatives were found to have a higher antiproliferative activity than cinnoline ones. The lowest IC50 values, below 20 μM, were obtained for quinoline and 2H-chromene-2,4(3H)-dione derivatives. Quinoline diamides were more efficient than cinnoline ones. Polyamine diimides containing phthalimide moieties demonstrated no inhibitory activities against melanoma cells. Preliminary studies of mechanism of action have shown that obtained derivatives were capable of quenching the fluorescence of ethidium bromide-DNA complex, indicating that they bound to ds-DNA in competition with ethidium bromide for binding sites. All the compounds were also subjected to preliminary in silico ADME screening by evaluating their theoretical drug-likeness and physicochemical properties using Discovery Studio 3.0 obtained from Accelrys. Compounds meeting the required ADME and drug-likeness criteria were selected.

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Polymerization activity of an alpha-like DNA polymerase requires a conserved 3'-5' exonuclease active site

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4786-4795.1991 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4786-4795

Author(s):

J S Gibbs ◽

K Weisshart ◽

P Digard ◽

A deBruynKops ◽

D M Knipe ◽

...

Keyword(s):

Dna Polymerase ◽

Active Site ◽

Dna Polymerases ◽

Polymerase Activity ◽

Cell Nuclei ◽

Dna Polymerase I ◽

Viral Dna ◽

Gene Encoding ◽

Simplex Virus ◽

Polymerase I

Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities. For Escherichia coli DNA polymerase I and its relatives, polymerase and exonuclease activities reside on distinct, separable domains of the same polypeptide. The catalytic subunits of the alpha-like DNA polymerase family share regions of sequence homology with the 3'-5' exonuclease active site of DNA polymerase I; in certain alpha-like DNA polymerases, these regions of homology have been shown to be important for exonuclease activity. This finding has led to the hypothesis that alpha-like DNA polymerases also contain a distinct 3'-5' exonuclease domain. We have introduced conservative substitutions into a 3'-5' exonuclease active site homology in the gene encoding herpes simplex virus DNA polymerase, an alpha-like polymerase. Two mutants were severely impaired for viral DNA replication and polymerase activity. The mutants were not detectably affected in the ability of the polymerase to interact with its accessory protein, UL42, or to colocalize in infected cell nuclei with the major viral DNA-binding protein, ICP8, suggesting that the mutation did not exert global effects on protein folding. The results raise the possibility that there is a fundamental difference between alpha-like DNA polymerases and E. coli DNA polymerase I, with less distinction between 3'-5' exonuclease and polymerase functions in alpha-like DNA polymerases.

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Composition and Function of Telomerase—A Polymerase Associated with the Origin of Eukaryotes

Biomolecules ◽

10.3390/biom10101425 ◽

2020 ◽

Vol 10 (10) ◽

pp. 1425

Author(s):

Petra Procházková Schrumpfová ◽

Jiří Fajkus

Keyword(s):

Dna Polymerase ◽

Dna Polymerases ◽

Telomerase Activity ◽

Structural Features ◽

Enzyme Complex ◽

Eukaryotic Evolution ◽

Linear Chromosomes ◽

And Function ◽

Phylogenetic Divergence ◽

Origin Of Eukaryotes

The canonical DNA polymerases involved in the replication of the genome are unable to fully replicate the physical ends of linear chromosomes, called telomeres. Chromosomal termini thus become shortened in each cell cycle. The maintenance of telomeres requires telomerase—a specific RNA-dependent DNA polymerase enzyme complex that carries its own RNA template and adds telomeric repeats to the ends of chromosomes using a reverse transcription mechanism. Both core subunits of telomerase—its catalytic telomerase reverse transcriptase (TERT) subunit and telomerase RNA (TR) component—were identified in quick succession in Tetrahymena more than 30 years ago. Since then, both telomerase subunits have been described in various organisms including yeasts, mammals, birds, reptiles and fish. Despite the fact that telomerase activity in plants was described 25 years ago and the TERT subunit four years later, a genuine plant TR has only recently been identified by our group. In this review, we focus on the structure, composition and function of telomerases. In addition, we discuss the origin and phylogenetic divergence of this unique RNA-dependent DNA polymerase as a witness of early eukaryotic evolution. Specifically, we discuss the latest information regarding the recently discovered TR component in plants, its conservation and its structural features.

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Double-stranded RNA bending by AU-tract sequences

Nucleic Acids Research ◽

10.1093/nar/gkaa1128 ◽

2020 ◽

Vol 48 (22) ◽

pp. 12917-12928

Author(s):

Alberto Marin-Gonzalez ◽

Clara Aicart-Ramos ◽

Mikel Marin-Baquero ◽

Alejandro Martín-González ◽

Maarit Suomalainen ◽

...

Keyword(s):

Nucleotide Sequence ◽

Double Helix ◽

Structural Features ◽

Sequence Motif ◽

Microscopy Imaging ◽

Rna Nanotechnology ◽

Sequence Dependent ◽

Structural Deformations ◽

Dynamics Simulations ◽

Dna Double Helix

Abstract Sequence-dependent structural deformations of the DNA double helix (dsDNA) have been extensively studied, where adenine tracts (A-tracts) provide a striking example for global bending in the molecule. However, in contrast to dsDNA, sequence-dependent structural features of dsRNA have received little attention. In this work, we demonstrate that the nucleotide sequence can induce a bend in a canonical Watson-Crick base-paired dsRNA helix. Using all-atom molecular dynamics simulations, we identified a sequence motif consisting of alternating adenines and uracils, or AU-tracts, that strongly bend the RNA double-helix. This finding was experimentally validated using atomic force microscopy imaging of dsRNA molecules designed to display macroscopic curvature via repetitions of phased AU-tract motifs. At the atomic level, this novel phenomenon originates from a localized compression of the dsRNA major groove and a large propeller twist at the position of the AU-tract. Moreover, the magnitude of the bending can be modulated by changing the length of the AU-tract. Altogether, our results demonstrate the possibility of modifying the dsRNA curvature by means of its nucleotide sequence, which may be exploited in the emerging field of RNA nanotechnology and might also constitute a natural mechanism for proteins to achieve recognition of specific dsRNA sequences.

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Polymerization activity of an alpha-like DNA polymerase requires a conserved 3'-5' exonuclease active site.

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4786 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4786-4795 ◽

Cited By ~ 25

Author(s):

J S Gibbs ◽

K Weisshart ◽

P Digard ◽

A deBruynKops ◽

D M Knipe ◽

...

Keyword(s):

Dna Polymerase ◽

Active Site ◽

Dna Polymerases ◽

Polymerase Activity ◽

Cell Nuclei ◽

Dna Polymerase I ◽

Viral Dna ◽

Gene Encoding ◽

Simplex Virus ◽

Polymerase I

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A polar filter in DNA polymerases prevents ribonucleotide incorporation

Nucleic Acids Research ◽

10.1093/nar/gkz792 ◽

2019 ◽

Vol 47 (20) ◽

pp. 10693-10705 ◽

Cited By ~ 4

Author(s):

Mary K Johnson ◽

Jithesh Kottur ◽

Deepak T Nair

Keyword(s):

Escherichia Coli ◽

Dna Polymerase ◽

Active Site ◽

Mycobacterium Smegmatis ◽

Dna Polymerases ◽

Drastic Reduction ◽

Equivalent Residue ◽

Stringent Selection ◽

Selection For ◽

Dna Polymerase Iv

Abstract The presence of ribonucleotides in DNA can lead to genomic instability and cellular lethality. To prevent adventitious rNTP incorporation, the majority of the DNA polymerases (dPols) possess a steric filter. The dPol named MsDpo4 (Mycobacterium smegmatis) naturally lacks this steric filter and hence is capable of rNTP addition. The introduction of the steric filter in MsDpo4 did not result in complete abrogation of the ability of this enzyme to incorporate ribonucleotides. In comparison, DNA polymerase IV (PolIV) from Escherichia coli exhibited stringent selection for deoxyribonucleotides. A comparison of MsDpo4 and PolIV led to the discovery of an additional polar filter responsible for sugar selectivity. Thr43 represents the filter in PolIV and this residue forms interactions with the incoming nucleotide to draw it closer to the enzyme surface. As a result, the 2’-OH in rNTPs will clash with the enzyme surface, and therefore ribonucleotides cannot be accommodated in the active site in a conformation compatible with productive catalysis. The substitution of the equivalent residue in MsDpo4–Cys47, with Thr led to a drastic reduction in the ability of the mycobacterial enzyme to incorporate rNTPs. Overall, our studies evince that the polar filter serves to prevent ribonucleotide incorporation by dPols.

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Genetic evidence for reconfiguration of DNA polymerase θ active site for error-free translesion synthesis in human cells

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.012816 ◽

2020 ◽

Vol 295 (18) ◽

pp. 5918-5927

Author(s):

Jung-Hoon Yoon ◽

Robert E. Johnson ◽

Louise Prakash ◽

Satya Prakash

Keyword(s):

Dna Polymerase ◽

Strong Evidence ◽

Active Site ◽

Dna Polymerases ◽

Translesion Synthesis ◽

Human Cells ◽

New Paradigm ◽

Tyrosine Residues ◽

Action Mechanisms ◽

Hoogsteen Base Pairing

The action mechanisms revealed by the biochemical and structural analyses of replicative and translesion synthesis (TLS) DNA polymerases (Pols) are retained in their cellular roles. In this regard, DNA polymerase θ differs from other Pols in that whereas purified Polθ misincorporates an A opposite 1,N6-ethenodeoxyadenosine (ϵdA) using an abasic-like mode, Polθ performs predominantly error-free TLS in human cells. To test the hypothesis that Polθ adopts a different mechanism for replicating through ϵdA in human cells than in the purified Pol, here we analyze the effects of mutations in the two highly conserved tyrosine residues, Tyr-2387 and Tyr-2391, in the Polθ active site. Our findings that these residues are indispensable for TLS by the purified Pol but are not required in human cells, as well as other findings, provide strong evidence that the Polθ active site is reconfigured in human cells to stabilize ϵdA in the syn conformation for Hoogsteen base pairing with the correct nucleotide. The evidence that a DNA polymerase can configure its active site entirely differently in human cells than in the purified Pol establishes a new paradigm for DNA polymerase function.

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