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PDBsum entry 2bqr
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References listed in PDB file
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Key reference
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Title
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Dna adduct bypass polymerization by sulfolobus solfataricus DNA polymerase dpo4: analysis and crystal structures of multiple base pair substitution and frameshift products with the adduct 1,N2-Ethenoguanine.
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Authors
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H.Zang,
A.K.Goodenough,
J.Y.Choi,
A.Irimia,
L.V.Loukachevitch,
I.D.Kozekov,
K.C.Angel,
C.J.Rizzo,
M.Egli,
F.P.Guengerich.
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Ref.
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J Biol Chem, 2005,
280,
29750-29764.
[DOI no: ]
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PubMed id
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Abstract
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1,N(2)-Etheno(epsilon)guanine is a mutagenic DNA lesion derived from lipid
oxidation products and also from some chemical carcinogens. Gel electrophoretic
analysis of the products of primer extension by Sulfolobus solfataricus P2 DNA
polymerase IV (Dpo4) indicated preferential incorporation of A opposite
3'-(1,N(2)-epsilon-G)TACT-5', among the four dNTPs tested individually. With the
template 3'-(1,N(2)-epsilon-G)CACT-5', both G and A were incorporated. When
primer extension was done in the presence of a mixture of all four dNTPs, high
pressure liquid chromatography-mass spectrometry analysis of the products
indicated that (opposite 3'-(1,N(2)-epsilon-G)CACT-5') the major product was
5'-GTGA-3' and the minor product was 5'-AGTGA-3'. With the template
3'-(1,N(2)-epsilon-G)TACT-5', the following four products were identified by
high pressure liquid chromatography-mass spectrometry: 5'-AATGA-3', 5'-ATTGA-3',
5'-ATGA-3', and 5'-TGA-3'. An x-ray crystal structure of Dpo4 was solved (2.1 A)
with a primer-template and A placed in the primer to be opposite the
1,N(2)-epsilon-G in the template 3'-(1,N(2)-epsilon-G)TACT 5'. The added A in
the primer was paired across the template T with classic Watson-Crick geometry.
Similar structures were observed in a ternary Dpo4-DNA-dATP complex and a
ternary Dpo4-DNA-ddATP complex, with d(d)ATP opposite the template T. A similar
structure was observed with a ddGTP adjacent to the primer and opposite the C
next to 1,N(2)-epsilon-G in 3'-(1,N(2)-epsilon-G)CACT-5'. We concluded that Dpo4
uses several mechanisms, including A incorporation opposite 1,N(2)-epsilon-G and
also a variation of dNTP-stabilized misalignment, to generate both base pair and
frameshift mutations.
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Figure 2.
FIG. 2. Extension of a 32P-labeled primer opposite template
adduct by pol T7^- and Dpo4 in the presence of single dNTPs. The
concentrations of each polymerase used are indicated. A, the
percentage of extension with 20 nM pol T7^- was 59, 4, 5, and 9%
for A, G, C, and T, respectively. B, the percentage of extension
with 100 nM Dpo4 was 23, 26, 8, and 11% for A, G., C, and T,
respectively.
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Figure 3.
FIG. 3. Extension of a 32P-labeled primer opposite the
template adduct by pol T7^- or Dpo4 in the presence of single
dNTPs. The concentrations of each polymerase used are indicated.
A, pol T7^-. B, Dpo4.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2005,
280,
29750-29764)
copyright 2005.
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