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PDBsum entry 6is7
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Transferase/DNA
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PDB id
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6is7
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PDB id:
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Transferase/DNA
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Title:
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Structure of 9n-i DNA polymerase incorporation with da in the active site
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Structure:
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DNA polymerase. Chain: a, b. Engineered: yes. Mutation: yes. DNA (5'-d(p Gp Cp Gp Gp Ap Cp Tp Gp Cp Tp Tp Ap Cp Cp A)- 3'). Chain: c, e, k, l. Engineered: yes. DNA (5'-
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Source:
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Thermococcus sp. 9on-7. Organism_taxid: 103799. Strain: 9on-7. Gene: pol, pola. Expressed in: escherichia coli 'bl21-gold(de3)plyss ag'. Expression_system_taxid: 866768. Synthetic: yes. Synthetic construct. Organism_taxid: 32630.
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Resolution:
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2.80Å
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R-factor:
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0.227
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R-free:
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0.276
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Authors:
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S.W.Linwu,M.Maestre-Reyna,M.D.Tsai,Y.H.Tu,W.H.Chang
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Key ref:
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S.W.LinWu
et al.
(2019).
Thermococcus sp. 9°N DNA polymerase exhibits 3'-esterase activity that can be harnessed for DNA sequencing.
Commun Biol,
2,
224.
PubMed id:
DOI:
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Date:
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15-Nov-18
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Release date:
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28-Aug-19
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PROCHECK
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Headers
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References
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Q56366
(DPOL_THES9) -
DNA polymerase from Thermococcus sp. (strain 9oN-7)
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Seq:
Struc:
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775 a.a.
757 a.a.*
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Key: |
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PfamA domain |
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Secondary structure |
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*
PDB and UniProt seqs differ
at 3 residue positions (black
crosses)
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G-C-G-G-A-C-T-G-C-T-T-A-C-C-A
15 bases
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A-C-T-G-G-T-A-A-G-C-A-G-T-C-C-G-C-G
18 bases
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G-C-G-G-A-C-T-G-C-T-T-A-C-C-A
15 bases
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A-C-T-G-G-T-A-A-G-C-A-G-T-C-C-G-C-G
18 bases
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A-A-G-C
4 bases
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C-T-T
3 bases
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T-G-C-T
4 bases
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Enzyme class:
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E.C.2.7.7.7
- DNA-directed Dna polymerase.
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Reaction:
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DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate
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DNA(n)
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+
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2'-deoxyribonucleoside 5'-triphosphate
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=
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DNA(n+1)
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+
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diphosphate
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Molecule diagrams generated from .mol files obtained from the
KEGG ftp site
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DOI no:
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Commun Biol
2:224
(2019)
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PubMed id:
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Thermococcus sp. 9°N DNA polymerase exhibits 3'-esterase activity that can be harnessed for DNA sequencing.
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S.W.LinWu,
Y.H.Tu,
T.Y.Tsai,
M.Maestre-Reyna,
M.S.Liu,
W.J.Wu,
J.Y.Huang,
H.W.Chi,
W.H.Chang,
C.F.Chiou,
A.H.Wang,
J.Lee,
M.D.Tsai.
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ABSTRACT
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It was reported in 1995 that T7 and Taq DNA polymerases possess 3'-esterase
activity, but without follow-up studies. Here we report that the 3'-esterase
activity is intrinsic to the Thermococcus sp. 9°N DNA polymerase, and
that it can be developed into a continuous method for DNA sequencing with dNTP
analogs carrying a 3'-ester with a fluorophore. We first show that 3'-esterified
dNTP can be incorporated into a template-primer DNA, and solve the crystal
structures of the reaction intermediates and products. Then we show that the
reaction can occur continuously, modulated by active site residues Tyr409 and
Asp542. Finally, we use 5'-FAM-labeled primer and esterified dNTP with a dye to
show that the reaction can proceed to ca. 450 base pairs, and that the
intermediates of many individual steps can be identified. The results
demonstrate the feasibility of a 3'-editing based DNA sequencing method that
could find practical applications after further optimization.
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