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PDBsum entry 2c56
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References listed in PDB file
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Key reference
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Title
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A comparative study of uracil-Dna glycosylases from human and herpes simplex virus type 1.
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Authors
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K.Krusong,
E.P.Carpenter,
S.R.Bellamy,
R.Savva,
G.S.Baldwin.
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Ref.
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J Biol Chem, 2006,
281,
4983-4992.
[DOI no: ]
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PubMed id
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Note In the PDB file this reference is
annotated as "TO BE PUBLISHED".
The citation details given above were identified by an automated
search of PubMed on title and author
names, giving a
percentage match of
95%.
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Abstract
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Uracil-DNA glycosylase (UNG) is the key enzyme responsible for initiation of
base excision repair. We have used both kinetic and binding assays for
comparative analysis of UNG enzymes from humans and herpes simplex virus type 1
(HSV-1). Steady-state fluorescence assays showed that hUNG has a much higher
specificity constant (k(cat)/K(m)) compared with the viral enzyme due to a lower
K(m). The binding of UNG to DNA was also studied using a catalytically inactive
mutant of UNG and non-cleavable substrate analogs (2'-deoxypseudouridine and
2'-alpha-fluoro-2'-deoxyuridine). Equilibrium DNA binding revealed that both
human and HSV-1 UNG enzymes bind to abasic DNA and both substrate analogs more
weakly than to uracil-containing DNA. Structure determination of HSV-1
D88N/H210N UNG in complex with uracil revealed detailed information on substrate
binding. Together, these results suggest that a significant proportion of the
binding energy is provided by specific interactions with the target uracil. The
kinetic parameters for human UNG indicate that it is likely to have activity
against both U.A and U.G mismatches in vivo. Weak binding to abasic DNA also
suggests that UNG activity is unlikely to be coupled to the subsequent common
steps of base excision repair.
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Figure 5.
FIGURE 5. Binding of the human wild-type and D145N/H268N
UNG enzymes to non-cleavable substrate analogs d  rd and
 -FdUrd. The binding of
hexachlorofluorescein-labeled oligonucleotides containing the
non-cleavable substrate analogs d rd and -FdUrd
was monitored using fluorescence polarization. A, the binding of
wild-type hUNG was measured with d rd (•) and -FdUrd (
 ).
Data are shown with the best fit to the binding equation with
the following values: d rd, K[d] = 4.4 ±
0.5 µM, A[D] = 0.041 ± 0.002, and A[D][E] = 0.17
± 0.003; and -FdUrd, K[d] = 6.3
± 0.6 µM, A[D] = 0.039 ± 0.002, and A[D][E]
= 0.13 ± 0.002. B, the binding of human D145N/H268N UNG
was measured with d rd (•) and -FdUrd (
).
Data are shown with the best fit to the binding equation with
the following values: d rd, K[d] = 3.2 ±
0.2 µM, A[D] = 0.038 ± 0.001, and A[D][E] = 0.14
± 0.001; and -FdUrd, K[d] = 2.2
± 0.2 µM, A[D] = 0.041 ± 0.003, and A[D][E]
= 0.17 ± 0.002.
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Figure 6.
FIGURE 6. Active site of HSV-1 D88N/H210N UNG. A, the amino
acid residues in the active site of HSV-1 D88N/H210N UNG (green,
carbons; blue, nitrogen; red, oxygens) are aligned with those of
the wild-type enzyme (purple). Electron density for HSV-1
D88N/H210N UNG is shown. B, HSV-1 D88N/H210N UNG with uridine
bound in the active site is aligned with the wild-type enzyme
with uracil bound in the active site (purple). Electron density
for HSV-1 D88N/H210N UNG is shown. C, hydrogen bond distances
between the residues in the HSV-1 D88N/H210N UNG active site and
the bound uracil are shown in red, whereas those in the active
site of the wild-type enzyme are shown in black.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2006,
281,
4983-4992)
copyright 2006.
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