 |
PDBsum entry 1pm0
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Transferase/DNA
|
PDB id
|
|
|
|
1pm0
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
Replication of a cis-Syn thymine dimer at atomic resolution.
|
|
|
Authors
|
|
H.Ling,
F.Boudsocq,
B.S.Plosky,
R.Woodgate,
W.Yang.
|
|
|
Ref.
|
|
Nature, 2003,
424,
1083-1087.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
Ultraviolet light damages DNA by catalysing covalent bond formation between
adjacent pyrimidines, generating cis-syn cyclobutane pyrimidine dimers (CPDs) as
the most common lesion. CPDs block DNA replication by high-fidelity DNA
polymerases, but they can be efficiently bypassed by the Y-family DNA polymerase
pol eta. Mutations in POLH encoding pol eta are implicated in nearly 20% of
xeroderma pigmentosum, a human disease characterized by extreme sensitivity to
sunlight and predisposition to skin cancer. Here we have determined two crystal
structures of Dpo4, an archaeal pol eta homologue, complexed with CPD-containing
DNA, where the 3' and 5' thymine of the CPD separately serves as a templating
base. The 3' thymine of the CPD forms a Watson-Crick base pair with the incoming
dideoxyATP, but the 5' thymine forms a Hoogsteen base pair with the dideoxyATP
in syn conformation. Dpo4 retains a similar tertiary structure, but each unusual
DNA structure is individually fitted into the active site for catalysis. A model
of the pol eta-CPD complex built from the crystal structures of Saccharomyces
cerevisiae apo-pol eta and the Dpo4-CPD complex suggests unique features that
allow pol eta to efficiently bypass CPDs.
|
|
|
|
|
|
|
|
Figure 1.
Figure 1: Replication of a CPD by Dpo4 in solution and crystals.
a, Extension of two primers (13 nucleotides each) paired with
undamaged or CPD-containing 18-nucleotide templates used in the
crystallization studies (TT-1, TT-2). Reactions were carried out
with 10 nM DNA substrate, 10 nM Dpo4 and 100  M
dATP at 37 °C for 2, 5, 10 or 20 min. b, Inhibition of primer
extension by ddATP. P indicates primer strand, and U and D
indicate undamaged and CPD-containing template, respectively.
The reactions took place for 30 min at 22 °C or 37 °C as
indicated. c, The active site of TT-1, where the 3' thymine of
the CPD (orange) is base-paired with ddATP (yellow). The
conserved residues interacting with ddATP and catalytic
carboxylates are highlighted. Tyr 10, which immobilizes the
finger domain by wedging between the palm and finger domains, is
shown in red. d, Replication at the 3' T of the CPD. The CPD and
the replicating and preceding base pairs of TT-1 are shown with
the F[o] - F[c] omit electron densities. e, Active site of TT-2,
where the 5' thymine of the CPD is base-paired with ddATP, f,
The replicating and two preceding base pairs of the TT-2 are
shown with the F[o] - F[c] omit electron densities. g, Stereo
view of the TT-1 (blue) and TT-2 (pink) active-site
superposition. The three catalytic carboxylates, two Ca^2+ ions,
the 3' nucleotide of the primer strand, and the incoming
nucleotide are shown in the ball-and-stick model.
|
|
Figure 2.
Figure 2: Structural comparison of the CPD complexed with Dpo4
and protein free. a, Structures of the CPD (orange) and
surrounding nucleic acids in TT-1 (blue), TT-2 (pink) and in the
absence of protein (yellow)19 are shown in the ball-and-stick
model after superposition of the CPDs. b, c, Ball-and-stick
presentations of base-pairing of the CPDs in TT-1 (b) and TT-2
(c). The phosphorus atoms are shown in purple, oxygen in red and
nitrogen in dark blue. The carbon atoms of the replicating base
pair are white, and others are light blue.
|
|
|
|
|
|
The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nature
(2003,
424,
1083-1087)
copyright 2003.
|
|
|
|
|
|
|
