scholarly journals Snapshots of a modified nucleotide moving through the confines of a DNA polymerase

2018 ◽  
Vol 115 (40) ◽  
pp. 9992-9997 ◽  
Author(s):  
Heike Maria Kropp ◽  
Simon Leonard Dürr ◽  
Christine Peter ◽  
Kay Diederichs ◽  
Andreas Marx
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Ternary Complexes ◽  
Structural Basis ◽  
Modified Nucleotides ◽  
Molecular Mechanics Calculations ◽  
Modified Dna ◽  
Substrate Properties ◽  
Dna Strand ◽  
Dna Substrate

DNA polymerases have evolved to process the four canonical nucleotides accurately. Nevertheless, these enzymes are also known to process modified nucleotides, which is the key to numerous core biotechnology applications. Processing of modified nucleotides includes incorporation of the modified nucleotide and postincorporation elongation to proceed with the synthesis of the nascent DNA strand. The structural basis for postincorporation elongation is currently unknown. We addressed this issue and successfully crystallized KlenTaq DNA polymerase in six closed ternary complexes containing the enzyme, the modified DNA substrate, and the incoming nucleotide. Each structure shows a high-resolution snapshot of the elongation of a modified primer, where the modification “moves” from the 3′-primer terminus upstream to the sixth nucleotide in the primer strand. Combining these data with quantum mechanics/molecular mechanics calculations and biochemical studies elucidates how the enzyme and the modified substrate mutually modulate their conformations without compromising the enzyme’s activity significantly. The study highlights the plasticity of the system as origin of the broad substrate properties of DNA polymerases and facilitates the design of improved systems.

KGF facilitates repair of radiation-induced DNA damage in alveolar epithelial cells

1997 ◽  
Vol 272 (6) ◽  
pp. L1174-L1180 ◽  
Author(s):  
M. Takeoka ◽  
W. F. Ward ◽  
H. Pollack ◽  
D. W. Kamp ◽  
R. J. Panos
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Epithelial Cells ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Dna Strand Breaks ◽  
Strand Breaks ◽  
Alveolar Epithelial ◽  
Radiation Induced ◽  
Dna Strand

Administration of exogenous keratinocyte growth factor (KGF) prevents or attenuates several forms of oxidant-mediated lung injury. Because DNA damage in epithelial cells is a component of radiation pneumotoxicity, we determined whether KGF ameliorated DNA strand breaks in irradiated A549 cells. Cells were exposed to 137Cs gamma rays, and DNA damage was measured by alkaline unwinding and ethidium bromide fluorescence after a 30-min recovery period. Radiation induced a dose-dependent increase in DNA strand breaks. The percentage of double-stranded DNA after exposure to 30 Gy increased from 44.6 +/- 3.5% in untreated control cells to 61.6 +/- 5.0% in cells cultured with 100 ng/ml KGF for 24 h (P < 0.05). No reduction in DNA damage occurred when the cells were cultured with KGF but maintained at 0 degree C during and after irradiation. The sparing effect of KGF on radiation-induced DNA damage was blocked by aphidicolin, an inhibitor of DNA polymerases-alpha, -delta, and -epsilon and by butylphenyl dGTP, which blocks DNA polymerase-alpha strongly and polymerases-delta and -epsilon less effectively. However, dideoxythymidine triphosphate, a specific inhibitor of DNA polymerase-beta, did not abrogate the KGF effect. Thus KGF increases DNA repair capacity in irradiated pulmonary epithelial cells, an effect mediated at least in part by DNA polymerases-alpha, -delta, and -epsilon. Enhancement of DNA repair capability after cell damage may be one mechanism by which KGF is able to ameliorate oxidant-mediated alveolar epithelial injury.

scholarly journals Mechanism of R-loop formation and conformational activation of Cas9

2021 ◽  
Author(s):  
Martin Pacesa ◽  
Martin Jinek
Keyword(s):  
Dna Cleavage ◽  
Structural Basis ◽  
Seed Region ◽  
Loop Formation ◽  
Base Pairs ◽  
Guide Rna ◽  
Target Strand ◽  
Target Dna ◽  
Dna Strand ◽  
Dna Substrate

Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage. The programmable activity of Cas9 has been widely utilized for genome editing applications. Despite extensive studies, the precise mechanism of target DNA binding and on-/off-target discrimination remains incompletely understood. Here we report cryo-EM structures of intermediate binding states of Streptococcus pyogenes Cas9 that reveal domain rearrangements induced by R-loop propagation and PAM-distal duplex positioning. At early stages of binding, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the PAM-distal duplex of the DNA substrate. Target hybridisation past the seed region positions the guide-target heteroduplex into the central binding channel and results in a conformational rearrangement of the REC lobe. Extension of the R-loop to 16 base pairs triggers the relocation of the HNH domain towards the target DNA strand in a catalytically incompetent conformation. The structures indicate that incomplete target strand pairing fails to induce the conformational displacements necessary for nuclease domain activation. Our results establish a structural basis for target DNA-dependent activation of Cas9 that advances our understanding of its off-target activity and will facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.

scholarly journals Mechanism of Inhibition of Vaccinia Virus DNA Polymerase by Cidofovir Diphosphate

2005 ◽  
Vol 49 (8) ◽  
pp. 3153-3162 ◽  
Author(s):  
Wendy C. Magee ◽  
Karl Y. Hostetler ◽  
David H. Evans
Keyword(s):  
Vaccinia Virus ◽  
Dna Polymerase ◽  
Viral Infections ◽  
Dna Polymerases ◽  
Antiviral Agent ◽  
Catalytic Efficiency ◽  
Inhibitory Mechanism ◽  
Reaction Products ◽  
Mechanism Of Inhibition ◽  
Dna Strand

ABSTRACT Cidofovir (CDV) is a broad-spectrum antiviral agent that has been approved for clinical use in the treatment of cytomegalovirus retinitis. It has also been used off label to treat a variety of other viral infections, including those caused by orf and molluscum contagiosum poxviruses. Because it is a dCMP analog, CDV is thought to act by inhibiting viral DNA polymerases. However, the details of the inhibitory mechanism are not well established and nothing is known about the mechanism by which the drug inhibits poxvirus DNA polymerases. To address this concern, we have studied the effect of the active intracellular metabolite of CDV, CDV diphosphate (CDVpp), on reactions catalyzed by vaccinia virus DNA polymerase. Using different primer-template pairs and purified vaccinia virus polymerase, we observed that CDV is incorporated into the growing DNA strand opposite template G's but the enzyme exhibits a lower catalytic efficiency compared with dCTP. CDV-terminated primers are also good substrates for the next deoxynucleoside monophosphate addition step, but these CDV + 1 reaction products are poor substrates for further rounds of synthesis. We also noted that although CDV can be excised from the primer 3′ terminus by the 3′-to-5′ proofreading exonuclease activity of vaccinia virus polymerase, DNAs bearing CDV as the penultimate 3′ residue are completely resistant to exonuclease attack. These results show that vaccinia virus DNA polymerase can use CDVpp as a dCTP analog, albeit one that slows the rate of primer extension. By inhibiting the activity of the proofreading exonuclease, the misincorporation of CDV could also promote error-prone DNA synthesis during poxvirus replication.

scholarly journals Involvement of the essential yeast DNA polymerases in induced gene conversion.

1999 ◽  
Vol 46 (4) ◽  
pp. 862-872 ◽  
Author(s):  
A Hałas ◽  
A Ciesielski ◽  
J Zuk
Keyword(s):  
Dna Synthesis ◽  
Gene Conversion ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dna Polymerase Alpha ◽  
Genes Encoding ◽  
Mitotic Gene Conversion ◽  
Dna Strand

In the yeast Saccharomyces cerevisiae three different DNA polymerases alpha, delta and epsilon are involved in DNA replication. DNA polymerase alpha is responsible for initiation of DNA synthesis and polymerases delta and epsilon are required for elongation of DNA strand during replication. DNA polymerases delta and epsilon are also involved in DNA repair. In this work we studied the role of these three DNA polymerases in the process of recombinational synthesis. Using thermo-sensitive heteroallelic mutants in genes encoding DNA polymerases we studied their role in the process of induced gene conversion. Mutant strains were treated with mutagens, incubated under permissive or restrictive conditions and the numbers of convertants obtained were compared. A very high difference in the number of convertants between restrictive and permissive conditions was observed for polymerases alpha and delta, which suggests that these two polymerases play an important role in DNA synthesis during mitotic gene conversion. Marginal dependence of gene conversion on the activity of polymerase epsilon indicates that this DNA polymerase may be involved in this process but rather as an auxiliary enzyme.

scholarly journals Structural basis for the synthesis of nucleobase modified DNA by Thermus aquaticus DNA polymerase

2010 ◽  
Vol 107 (50) ◽  
pp. 21327-21331 ◽  
Author(s):  
S. Obeid ◽  
A. Baccaro ◽  
W. Welte ◽  
K. Diederichs ◽  
A. Marx
Keyword(s):  
Dna Polymerase ◽  
Structural Basis ◽  
Thermus Aquaticus ◽  
Modified Dna

scholarly journals Structural and kinetic insights into binding and incorporation of L-nucleotide analogs by a Y-family DNA polymerase

2014 ◽  
Vol 42 (15) ◽  
pp. 9984-9995 ◽  
Author(s):  
Vineet Gaur ◽  
Rajan Vyas ◽  
Jason D. Fowler ◽  
Georgia Efthimiopoulos ◽  
Joy Y. Feng ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Building Blocks ◽  
Structural Basis ◽  
Binding Modes ◽  
Nucleotide Analogs ◽  
Reverse Transcriptases ◽  
Sugar Ring ◽  
Sugar Puckering ◽  
Y Family

AbstractConsidering that all natural nucleotides (D-dNTPs) and the building blocks (D-dNMPs) of DNA chains possess D-stereochemistry, DNA polymerases and reverse transcriptases (RTs) likely possess strongD-stereoselectivity by preferably binding and incorporating D-dNTPs over unnatural L-dNTPs during DNA synthesis. Surprisingly, a structural basis for the discrimination against L-dNTPs by DNA polymerases or RTs has not been established although L-deoxycytidine analogs (lamivudine and emtricitabine) and L-thymidine (telbivudine) have been widely used as antiviral drugs for years. Here we report seven high-resolution ternary crystal structures of a prototype Y-family DNA polymerase, DNA, and D-dCTP, D-dCDP, L-dCDP, or the diphosphates and triphosphates of lamivudine and emtricitabine. These structures reveal that relative to D-dCTP, each of these L-nucleotides has its sugar ring rotated by 180° with an unusual O4′-endo sugar puckering and exhibits multiple triphosphate-binding conformations within the active site of the polymerase. Such rare binding modes significantly decrease the incorporation rates and efficiencies of these L-nucleotides catalyzed by the polymerase.

scholarly journals Conformational dynamics during misincorporation and mismatch extension defined using a DNA polymerase with a fluorescent artificial amino acid

2021 ◽  
Author(s):  
Tyler L Dangerfield ◽  
Serdal Kirmizialtin ◽  
Kenneth A. Johnson
Keyword(s):  
Amino Acid ◽  
Dna Polymerase ◽  
Active Site ◽  
Dna Polymerases ◽  
Conformational Dynamics ◽  
Unnatural Amino Acid ◽  
Structural Basis ◽  
High Fidelity ◽  
Active Site Residues ◽  
Kinetic Measurements

High-fidelity DNA polymerases select the correct nucleotide over the structurally similar incorrect nucleotides with extremely high specificity while maintaining fast rates of incorporation. Previous analysis revealed the conformational dynamics and complete kinetic pathway governing correct nucleotide incorporation using a high-fidelity DNA polymerase variant containing a fluorescent unnatural amino acid. Here we extend this analysis to investigate the kinetics of nucleotide misincorporation and mismatch extension. We report the specificity constants for all possible misincorporations and characterize the conformational dynamics of the enzyme during misincorporation and mismatch extension. We present free energy profiles based on the kinetic measurements and discuss the effect of different steps on specificity. During mismatch incorporation and subsequent extension (with the correct nucleotide), the rates of the conformational change and chemistry are both greatly reduced. The nucleotide dissociation rate, however, increases to greatly exceed the rate of chemistry. To investigate the structural basis for discrimination against mismatched nucleotides, we performed all atom molecular dynamics simulations on complexes with either the correct or mismatched nucleotide bound at the polymerase active site. We show that the closed form of the enzyme with a mismatch bound is greatly destabilized due to weaker interactions with active site residues, non-ideal base pairing, and a large increase in the distance from the 3′-OH group of the primer strand to the α-phosphate of the incoming nucleotide, explaining the reduced rates of misincorporation. The observed kinetic and structural mechanisms governing nucleotide misincorporation reveal the general principles likely applicable to other high fidelity DNA polymerases.

scholarly journals Bypass of the Major Alkylative DNA Lesion by Human DNA Polymerase η

Molecules ◽  
2019 ◽  
Vol 24 (21) ◽  
pp. 3928 ◽  
Author(s):  
Myong-Chul Koag ◽  
Hunmin Jung ◽  
Yi Kou ◽  
Seongmin Lee
Keyword(s):  
Dna Polymerase ◽  
Base Pair ◽  
Alkylating Agents ◽  
Dna Polymerases ◽  
Translesion Synthesis ◽  
Catalytic Site ◽  
Structural Basis ◽  
Dna Lesion ◽  
Human Dna ◽  
Wide Range

A wide range of endogenous and exogenous alkylating agents attack DNA to generate various alkylation adducts. N7-methyl-2-deoxyguanosine (Fm7dG) is the most abundant alkylative DNA lesion. If not repaired, Fm7dG can undergo spontaneous depurination, imidazole ring-opening, or bypass by translesion synthesis DNA polymerases. Human DNA polymerase η (polη) efficiently catalyzes across Fm7dG in vitro, but its structural basis is unknown. Herein, we report a crystal structure of polη in complex with templating Fm7dG and an incoming nonhydrolyzable dCTP analog, where a 2′-fluorine-mediated transition destabilization approach was used to prevent the spontaneous depurination of Fm7dG. The structure showed that polη readily accommodated the Fm7dG:dCTP base pair with little conformational change of protein and DNA. In the catalytic site, Fm7dG and dCTP formed three hydrogen bonds with a Watson–Crick geometry, indicating that the major keto tautomer of Fm7dG is involved in base pairing. The polη-Fm7dG:dCTP structure was essentially identical to the corresponding undamaged structure, which explained the efficient bypass of the major methylated lesion. Overall, the first structure of translesion synthesis DNA polymerase bypassing Fm7dG suggests that in the catalytic site of Y-family DNA polymerases, small N7-alkylguanine adducts may be well tolerated and form the canonical Watson–Crick base pair with dCTP through their keto tautomers.

scholarly journals Structural Basis for the KlenTaq DNA Polymerase Catalysed Incorporation of Alkene- versus Alkyne-Modified Nucleotides

2017 ◽  
Vol 23 (9) ◽  
pp. 2109-2118 ◽  
Author(s):  
Audrey Hottin ◽  
Karin Betz ◽  
Kay Diederichs ◽  
Andreas Marx
Keyword(s):  
Dna Polymerase ◽  
Structural Basis ◽  
Modified Nucleotides
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