Download Regulation of Nucleotide Excision Repair: UV-DDB

Nina Kaczmarek, Jia Fei and Hanspeter Nägeli
Institute of Pharmacology and Toxicology
University of Zürich-Vetsuisse, CH-8057 Zürich
Regulation of Nucleotide Excision Repair: UV-DDB-dependent
Prioritization of Damage Recognition in intranucleosomal DNA INTRODUCTION
A
The UV-damaged DNA-binding (UV-DDB) and XPC-RAD23B complexes are the
initial sensors of UV lesions that trigger Nuleotide Excision Repair (NER) activity
throughout the genome. UV-DDB is a heterodimer: DDB1 associates with the
CUL4A ubiquitin ligase (Fig 1A) whereas DDB2 binds avidly to UV-irradiated
DNA. Previous studies demonstrated that UV-DDB translocates to chromatin
immediately after UV irradiation and that this UV lesion recognition factor binds
with highest affinity to 6-4PP. Since the identification of UV-DDB as a DNA
damage recognition factor, this complex has been the subject of intense
scrutiny, but its role in DNA repair remained enigmatic. Because 6-4PPs are
formed preferentially in the linker DNA that separates nucleosome core
particles, the basic unit of chromatin, we tested the hypothesis that UV-DDB
accumulates mainly at internucleosomal sites. Interestingly, lower eukaryotes
lack DDB2, indicating that this coordinator of DNA repair becomes critical in
vertebrates, where a large genome involves hierarchical levels of chromatin
compaction (Fig 1B).
B
C
C
Baylin et al. (2007)
Scrima et al. (2008)
RESULTS
Translocation of UV-DDB to Internucleosomal DNA in UV-irradiated cells
A Lysis of 6 x 106 cells Centrifuga?on A B with NP-­‐40 buffer Supernatant 1 = Free (non-­‐chroma?n bound) proteins (500 µl) DDB2-XPC interactions stimulated by DNA damage
A Insoluble (40%)! Soluble (40%)!
Free (8%)!
B C C Mnase diges?on of the pellet in CS buffer Centrifuga?on Supernatant 2 = Solubilizable chroma?n frac?on (50 µl) Insoluble chroma?n frac?on (in 80 µl denaturing buffer) C Insoluble (40%)! Soluble (40%)! Free (8%)!
D E 250
150
100
Fig 2 The distribution of NER factors and histones
in chromatin was analyzed by MNase digestion
(4 U/µl) (A) followed by immunoblotting in HeLa
(B) and in p53-proficient U2OS cells (C). The
numbers in parenthesis indicate the proportion of
each fraction loaded onto the gel.
A A A Insoluble (40%)!
B A Dynamic DDB2-XPC handover
Bleach
Ubiquitin-dependent XPC Partitoning in Chromatin
Fig 3. Early accumulation of XPC at
solubilizable internucleosomal sites is
ubiquitin-independent
(A),
whereas
retention of XPC at these sites depends
on
UV-DDBCUL4A
mediated
ubiquitination (B-C). The numbers in
parenthesis indicate the proportion of
each fraction loaded onto the gel.
Soluble (40%)!
Prebleach
Postbleach
B B C Soluble (67%
)!
(67)
Insoluble (30%
)!
(40)
Summary
Ubiquitin-independent XPC recruitment by DDB2
A A B C αGAPDH Fig 4. Ubiquitination of endogenous XPC (125kDa) but not of XPC-GFP (150kDa) (A). XPC-GFP relocation in CHO cells to
irradiated areas (B). XPC-GFP relocation to UV lesions stimulated by co-expression of DDB2-RFP. GFP signals at UV lesion
spots (N=30) were quantified, normalized against the nuclear background and expressed as a percentage of controls (XPC
alone) (C). Fig 5. Domain structure of
human XPC (A). Recruitment
of XPC-GFP truncates (B) and
deletions (C) to UV-lesions.
Fluorescence
spots
colocalizing with UV lesions
(N=30) were normalized and
expressed as a percentage of
control values (full-length XPC
alone). Interaction between
DDB2 and XPC428-633-GST
inhibited by a 15-mer DNA
duplex
(120
pmol)
(D)
Interactions between DDB2
and
XPC607-741-GST
are
stimulated by a 15-mer DNA
duplex (top panel: 15-60 pmol;
bottom panel: 120 pmol).
Fig 6. The DDB2 subunit of UV-DDB
interacts weakly with the TGD motif of
XPC and, in the proximity of UV lesions,
exerts a bimodal action. By transient
contacts with the BHD1 motif of XPC, the
DDB2 subunit facilitates a β-hairpin
insertion that locally unwinds the DNA
double helix. This direct function is
required across the whole genome for the
excision of CPDs that, on their own,
induce minimal distortions of the DNA
duplex and, hence, are not directly
recognizable by XPC alone. The UV-DDB
accumulation at internucleosomal sites
leads to polyubiquitination of the XPC
partner thus promoting its retention. The
implementation of this ubiquitin code is
mainly required for the fast initial excision
of 6-4PPs, which are enriched in
internucleosomal DNA and directly
recognizable by XPC protein.
C Fig 6. FRAP (Fluorescence
recovery
after
Photobleaching) on local damage
analysis (A). Dissociation
of XPC-GFP from UV
lesions in CHO cells
measured by FRAP-LD
(N=15; ±s.e.m.). Half-lives
were estimated from each
fluorescence
recovery
curve (B). DDB2 is unable
to stabilize the ΔHairpin
deletion at UV lesions in
FRAP-LD assays (N=15)
(C).
Fast excision from
internucleosomal sites