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Circadian Control of Nucleotide Excision Repair
Tae‐Hong Kang
University of North Carolina at Chapel Hill
Dept. of Biochemistry & Biophysics
Aziz Sancar Lab
Young Investigator Talks in DNA Repair Videoconference May 18, 2010
Circadian (Daily) Oscillation of Biological Processes
Maury et al. Circ Res (2010) 106, 447‐462
www.learner.org/jnorth/images
circa, "around," and diem or dies, “a day"
Circadian (Daily) Rhythm
Nature (2002) 418, 935‐941
Hierarchy of Mammalian Circadian Timing System
SCN: suprachiasmatic nucleus
Molecular Clock
Sancar A Nat Struct Mol Biol (2008) 15, 234‐235
Loss of Rhythmic Behavior in Clock‐deficient Mice
Nature (1999) 398, 627‐630
Dark Light Dark LightLight Dark Light Dark LightLight
Actogram: Wheel running activityClock
Bmal1Cry
Per
Capacity of Molecular Clock
Sancar A Nat Struct Mol Biol (2008) 15, 234‐235 Sancar A Nat Struct Mol Biol (2008) 15, 234‐235
~ 10% of transcriptome/tissue
Circadian Control of Cell‐division Cycles
Nat Rev Genet (2008) 9, 764‐75
PtCl
ClH3N
H3N
Chemotherapeutic agents (Cisplatin)
XPA
RPA
TFIIH
XPC
XPA
RPAXPCTFIIH
XPA
RPA
TFIIH XPA
RPA
TFIIH
3’ Incision5’ Incision
PIC1
PIC2 PIC3XPGXPG
Pol δ/ε
Ligase
Excision product
XPF
Nucleotide Excision Repair
Kang & Sancar Cell Cycle (2009) 8, 1665‐1667
Xeroderma pigmentosum (XP)
Google Image
Excision Products(24‐32 nt)
Nucleotide Excision Repair(Dual incisions)
Circular (2.9 kb) or Linear (140 bp)
5’3’ 5’
3’
5’3’ 5’
3’
5’ 3’
3’ 5’
DNA Excision Repair Activity Assay
Sacrifice mice every 4h for 24h
Tissue Collection
Homogenization
Cell‐free extract
(Manley Method)
32P
Pt_GTG adduct
Circadian Oscillation of NER Activity
Rel
ativ
e N
ER a
ctiv
ity0
20
40
60
80
100
02 06 10 14 18 22 :ZT(09) (13) (17) (21) (01) (05) :EST
mag
nific
atio
n
XPG
XPF
XPD
XPB
XPA
XPC
RPA 70
ZT06ZT18
Circadian Oscillation of XPA
Loss of Circadian Oscillation of XPA expression and NER Activity in Clock‐deficient mouse
Degradation of XPA by the proteasome
(h)
XPA
1 3 6 1 3 6CHX MG132
ACTIN
XPA
ACTINA
549
cell
Rat
1 ce
ll
0
mRNA
DNAXPA
Protein
Degradation
CHX
MG132
XPA
Interaction of XPA and ubiquitin E3 ligase HERC2
250
150
100
75
50
35
RAD1 XPA : IP
XPA
HERC2
RPA1C
ontr
ol
Flag
-XPA
Con
trol
Flag
-XPA
Con
trol
Flag
-XPA
Input Unbound Bound
IP: αFlag
HERC2
XPA
RPA70
MDM2
RPA32HERC1
HERC XPA Merge
HERC1
HERC2
Interaction of XPA and ubiquitin E3 ligase HERC2
HERC2 is an E3 Ub‐ligase for XPA
Mock
Augmentation of NER activity by HERC2 down‐regulation
CTRL HERC2 CTRL HERC2
0
2
4
8
12
24
Mock
:siRNA
Time (h) a
fter UV
20 J/m2 10 J/m2 :UV
α‐CPD
CTRL HERC2 CTRL HERC2
30 μM 10 μM
0
2
4
8
12
Mock
:siRNA
:Cisplatin
Time (h) a
fter Cisplatin
α‐Pt_GG
Nucleotide excision repair
Day Night
XPA
Day Night
Day Night
Day Night
Circadian Clock
Testis, CryDKO
Conclusion
HERC2
?
Liver, Brain, Kidney …
[XPA
]
[XPA
]
Excision
rep
air
Excision
rep
air
Time of the day
Time of the day
Time of the day
Time of the day
Kang et al. PNAS (2010) 107, 4890‐4895Kang et al. PNAS (2009) 106, 2864‐2867
Timing for Cisplatin treatment
Chrono‐Chemotherapy: Circadian Timing for Cancer Chemotherapy
XPA expression (NER activity) in
mouse tissue
Time (clock)
healthy tumor
?
Acknowledgements
Aziz Sancar
Joyce Reardon
Laura Lindsey‐Boltz
Michael Kemp
University of North Carolina at Chapel HillDept. of Biochemistry & Biophysics
Aziz Sancar Lab
Circadian Control of DNA Repair