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PDBsum entry 2pie
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References listed in PDB file
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Key reference
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Title
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Rnf8 transduces the DNA-Damage signal via histone ubiquitylation and checkpoint protein assembly.
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Authors
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M.S.Huen,
R.Grant,
I.Manke,
K.Minn,
X.Yu,
M.B.Yaffe,
J.Chen.
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Ref.
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Cell, 2007,
131,
901-914.
[DOI no: ]
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PubMed id
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Abstract
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DNA-damage signaling utilizes a multitude of posttranslational modifiers as
molecular switches to regulate cell-cycle checkpoints, DNA repair, cellular
senescence, and apoptosis. Here we show that RNF8, a FHA/RING domain-containing
protein, plays a critical role in the early DNA-damage response. We have solved
the X-ray crystal structure of the FHA domain structure at 1.35 A. We have shown
that RNF8 facilitates the accumulation of checkpoint mediator proteins BRCA1 and
53BP1 to the damaged chromatin, on one hand through the phospho-dependent FHA
domain-mediated binding of RNF8 to MDC1, on the other hand via its role in
ubiquitylating H2AX and possibly other substrates at damage sites. Moreover,
RNF8-depleted cells displayed a defective G2/M checkpoint and increased IR
sensitivity. Together, our study implicates RNF8 as a novel
DNA-damage-responsive protein that integrates protein phosphorylation and
ubiquitylation signaling and plays a critical role in the cellular response to
genotoxic stress.
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Figure 2.
Figure 2. Structural Basis for Phosphorylation-Dependent
Binding by RNF8 FHA Domain
(A) Amino acid selectivity
values for the RNF8 FHA domain determined using the
phosphothreonine-oriented degenerate peptide library
MAXXXX-pT-XXXXAKKK, where X indicates all amino acids except
Cys. Values ≥ 1.4 indicate moderate selection; values ≥ 2.0
indicate strong selection.
(B) Cartoon representation of
the RNF8 FHA domain bound to the optimal phosphopeptide
ELKpTERY.
(C) stereo view of the phosphopeptide-binding
surface.
(D) Closeup of the phosphate-binding pocket, with
2Fo-Fc density map contoured at 2σ. A bound water molecule is
evident in the upper center.
(E–G) Molecular interaction
surfaces of the RNF8:phosphopeptide complex, the MDC1 tandem
BRCT domain:γ-H[2]AX phosphopeptide complex, and the Rad53
FHA1:LEVpTEAD phosphopeptide complex. Peptide surfaces are
contoured in salmon, protein surfaces are contoured in lime. In
the RNF8 FHA domain (E), selection for Tyr over Phe in the +3
position likely results from a water-mediated contact between
the Tyr hydroxyl and the backbone nitrogen of Leu57. In the
Rad53 FHA1 structure (G), an Arg residue from the FHA domain
occupies the equivalent position as the peptide +3 Tyr in the
RNF8 structure (dashed line).
(H) Divergence in the
phospho-amino acid +3 binding surfaces of the FHA domains of
RNF8 and Rad53. The Cα traces of the FHA domains of the RNF8
FHA domain:phosphopeptide complex and the Rad53 FHA1
domain:phosphopeptide complex were optimally aligned. The
phosphopeptide +3 interacting region is shown in cartoon
representation, with the RNF8 FHA domain shaded blue and its
bound phosphopeptide shaded cyan, while the Rad53 FHA1 domain is
shaded yellow and its bound phosphopeptide is shaded green. The
+3 Tyr residue in the RNF8 optimal phosphopeptide and the +3 Asp
in the Rad53 FHA1 optimal phosphopeptide are shown in stick
representation. Note that the +3 Tyr binding site in RNF8 is
occluded in the Rad53 FHA domain by an Arg residue that mediates
selection for Asp in the +3 position.
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Figure 6.
Figure 6. RNF8 Is Required for H2AX Ubiquitylation following
DNA Damage
(A) H2AX is ubiquitylated in vivo. 293T cells
were transiently transfected with plasmids encoding myc-tagged
ubiquitin with or without plasmids encoding SBP-Flag-H2AX.
Immunoprecipitation and immunoblotting were carried out using
indicated antibodies. Black arrow indicates doubly ubiquitylated
species of H2AX, while gray arrow indicates monoubiquitinated
H2AX. Multiple-ubiquitinated H2AX species are also pointed out.
(B) HeLa cells stably expressing HA-tagged H2AX were
transfected with control siRNA or RNF8 siRNA were treated with
10 Gy or left untreated. Cells were harvested 1 hr
post-irradiation. Cell lysates were prepared, separated by
SDS-PAGE, and blotted with indicated antibodies.
(C) HeLa
cells transfected with control siRNA or RNF8 siRNA were treated
as described in (B) and immunoblotting experiments were carried
out using indicated antibodies.
(D) IR-induced H2AX
ubiquitylation in H2AX^+/+ and H2AX^−/− MEFs. Cell lysates
prepared from WT or H2AX^−/− cells before and after
irradiation were immunoblotted with anti-H2AX and anti-pH2AX
antibodies.
(E) IR-induced H2AX ubiquitylation requires
H2AX phosphorylation. H2AX-deficient MEFs stably expressing
HA-tagged H2AX or S139A mutant of H2AX were treated with 0 Gy or
10 Gy and immunoblotting was performed using indicated
antibodies.
(F) IR-induced H2AX ubiquitylation requires
RNF8 FHA and RING domains. Experiments were carried out as that
described in Figure 4C. Immunoblotting experiments were
conducted with antibodies as indicated. Arrow indicates
ubiquitylated species of H2AX that only appear after radiation
in cells expressing WT RNF8.
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The above figures are
reprinted
from an Open Access publication published by Cell Press:
Cell
(2007,
131,
901-914)
copyright 2007.
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