Binding of Mn-deoxyribonucleoside triphosphates to the active site of the DNA polymerase of bacteriophage T7

Divalent metal ions are crucial as cofactors for a variety of intracellular enzymatic activities. Mg2+, as an example, mediates binding of deoxyribonucleoside 5′-triphosphates followed by their hydrolysis in the active site of DNA polymerase. It is difficult to study the binding of Mg2+ to an active site because Mg2+ is spectroscopically silent and Mg2+ binds with low affinity to the active site of an enzyme. Therefore, we substituted Mg2+ with Mn2+:Mn2+ that is not only visible spectroscopically but also provides full activity of the DNA polymerase of bacteriophage T7. In order to demonstrate that the majority of Mn2+ is bound to the enzyme, we have applied site-directed titration analysis of T7 DNA polymerase using X-ray near edge spectroscopy. Here we show how X-ray near edge spectroscopy can be used to distinguish between signal originating from Mn2+ that is free in solution and Mn2+ bound to the active site of T7 DNA polymerase. This method can be applied to other enzymes that use divalent metal ions as a cofactor.

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Structure of the endonuclease IV homologue fromThermotoga maritimain the presence of active-site divalent metal ions

Acta Crystallographica Section F Structural Biology and Crystallization Communications ◽

10.1107/s1744309110028575 ◽

2010 ◽

Vol 66 (9) ◽

pp. 1003-1012 ◽

Cited By ~ 5

Author(s):

Stephen J. Tomanicek ◽

Ronny C. Hughes ◽

Joseph D. Ng ◽

Leighton Coates

Keyword(s):

Metal Ions ◽

Active Site ◽

Excision Repair ◽

Divalent Metal ◽

Hydroxyl Group ◽

Divalent Metal Ions ◽

Ap Endonuclease ◽

Phosphodiester Bond ◽

Dna Base ◽

Ap Site

The most frequent lesion in DNA is at apurinic/apyrimidinic (AP) sites resulting from DNA-base losses. These AP-site lesions can stall DNA replication and lead to genome instability if left unrepaired. The AP endonucleases are an important class of enzymes that are involved in the repair of AP-site intermediates during damage-general DNA base-excision repair pathways. These enzymes hydrolytically cleave the 5′-phosphodiester bond at an AP site to generate a free 3′-hydroxyl group and a 5′-terminal sugar phosphate using their AP nuclease activity. Specifically,Thermotoga maritimaendonuclease IV is a member of the second conserved AP endonuclease family that includesEscherichia coliendonuclease IV, which is the archetype of the AP endonuclease superfamily. In order to more fully characterize the AP endonuclease family of enzymes, two X-ray crystal structures of theT. maritimaendonuclease IV homologue were determined in the presence of divalent metal ions bound in the active-site region. These structures of theT. maritimaendonuclease IV homologue further revealed the use of the TIM-barrel fold and the trinuclear metal binding site as important highly conserved structural elements that are involved in DNA-binding and AP-site repair processes in the AP endonuclease superfamily.

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Divalent metal ions influence catalysis and active-site accessibility in the camp-dependent protein kinase

Protein Science ◽

10.1002/pro.5560021217 ◽

1993 ◽

Vol 2 (12) ◽

pp. 2177-2186 ◽

Cited By ~ 42

Author(s):

Joseph A. Adams ◽

Susan S. Taylor

Keyword(s):

Protein Kinase ◽

Metal Ions ◽

Active Site ◽

Divalent Metal ◽

Divalent Metal Ions ◽

Dependent Protein Kinase ◽

Camp Dependent Protein Kinase ◽

Dependent Protein

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Structural and Binding Analysis of Pyrimidinol Carboxylic Acid andN-Hydroxy Quinazolinedione HIV-1 RNase H Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01594-10 ◽

2011 ◽

Vol 55 (6) ◽

pp. 2905-2915 ◽

Cited By ~ 58

Author(s):

Eric B. Lansdon ◽

Qi Liu ◽

Stephanie A. Leavitt ◽

Mini Balakrishnan ◽

Jason K. Perry ◽

...

Keyword(s):

Carboxylic Acid ◽

Metal Ions ◽

Active Site ◽

Water Environment ◽

Divalent Metal ◽

Rnase H ◽

Accurate Assessment ◽

Divalent Metal Ions ◽

Substrate State ◽

Hiv 1

ABSTRACTHIV-1 RNase H breaks down the intermediate RNA-DNA hybrids during reverse transcription, requiring two divalent metal ions for activity. Pyrimidinol carboxylic acid andN-hydroxy quinazolinedione inhibitors were designed to coordinate the two metal ions in the active site of RNase H. High-resolution (1.4 Å to 2.1 Å) crystal structures were determined with the isolated RNase H domain and reverse transcriptase (RT), which permit accurate assessment of the metal and water environment at the active site. The geometry of the metal coordination suggests that the inhibitors mimic a substrate state prior to phosphodiester catalysis. Surface plasmon resonance studies confirm metal-dependent binding to RNase H and demonstrate that the inhibitors do not bind at the polymerase active site of RT. Additional evaluation of the RNase H site reveals an open protein surface with few additional interactions to optimize active-site inhibitors.

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Structure of New Binary and Ternary DNA Polymerase Complexes From Bacteriophage RB69

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.704813 ◽

2021 ◽

Vol 8 ◽

Author(s):

Jongseo Park ◽

Hyung-Seop Youn ◽

Jun Yop An ◽

Youngjin Lee ◽

Soo Hyun Eom ◽

...

Keyword(s):

Metal Ions ◽

Dna Polymerase ◽

Metal Ion ◽

Ternary Complex ◽

Critical Role ◽

Divalent Metal ◽

Binary Complex ◽

Divalent Metal Ions ◽

Divalent Metal Ion ◽

Initial Binding

DNA polymerase plays a critical role in passing the genetic information of any living organism to its offspring. DNA polymerase from enterobacteria phage RB69 (RB69pol) has both polymerization and exonuclease activities and has been extensively studied as a model system for B-family DNA polymerases. Many binary and ternary complex structures of RB69pol are known, and they all contain a single polymerase-primer/template (P/T) DNA complex. Here, we report a crystal structure of the exonuclease-deficient RB69pol with the P/T duplex in a dimeric form at a resolution of 2.2 Å. The structure includes one new closed ternary complex with a single divalent metal ion bound and one new open binary complex in the pre-insertion state with a vacant dNTP-binding pocket. These complexes suggest that initial binding of the correct dNTP in the open state is much weaker than expected and that initial binding of the second divalent metal ion in the closed state is also much weaker than measured. Additional conformational changes are required to convert these complexes to high-affinity states. Thus, the measured affinities for the correct incoming dNTP and divalent metal ions are average values from many conformationally distinctive states. Our structure provides new insights into the order of the complex assembly involving two divalent metal ions. The biological relevance of specific interactions observed between one RB69pol and the P/T duplex bound to the second RB69pol observed within this dimeric complex is discussed.

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