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