2.  Crosstalk between histone modifications during the DNA damage response ◦ Histone ◦ Histone modification ◦ DNA damage ◦ DNA damage response

3.  The componential proteins unit of nucleosome  Histone Octamer ◦ H2A, H2B, H3, H4 ◦ Nature Reviews Cancer 1, 194-202 (December 2001) | doi:10.1038/35106079

4.  Protein can be modified!  Acetylation, Phosphorylation, Ubiquitination, Mehtylation, Etc  Histone octamer can be also modified Histone is also protein! Nature Reviews Cancer 1, 194-202 (December 2001) | doi:10.1038/35106079

5.  Double Strand Break(DSB)  Single Strand Break(SSB)  Thymine Dimer by UV

6.  The most deleterious forms of DNA damage ◦ In most case of SSB, there are templates

7.  Repair  Death(Apoptosis)  Aging

8.  Base excision repair(BER)  Nucleotide excision repair(NER)  DSB repair ◦ Homologous recombination(HR) ◦ Non-homologous end-joining(NHEJ)

9. Peterson C L, Côté J Genes Dev. 2004;18:602-616

10.  -Ray

11.  Repair Or Die?  To determine this, Body has to know about DNA damage  How can detect DNA damage? Especially, DSB

12. H2A variant form 2~25 % population in tissue Conserved motif : SQEY Especially,‘S’ is very critical factor in Damage Response  H2AX is S139 phosphorylated form When DSB occurs,  H2AX appears -> DSB marker

13.  Doctor : DNA damage response proteins  Amplification of  H2AX  Formation of foci

14.  MRN complex recognizes DSB site, and binds to DNA end.  And recruits activated ATM  ATM phosphoryates H2AX(it called  H2AX)  NBS1 recruits MDC1 to  H2AX  Amplification of  H2AX Recognition and signal amplification of DNA damage

15. Michael B. Kastan, Cancer Res 2008;6(4):517–24 ATM also phosphorylates MDC1, 53BP1, BRCA1

16.  I’m in trouble!  Call doctor! Real Doctor makes holes to reach damaged site How about DNA repair?

17.  To expose damaged DNA, chromatin must change  -> Loosing!

18.  Acetylation  Ubiquitination  Methylation

19.  Generally when acetylated in histone  -> Loosing  And when deacetylated in histone  -> Packing

20.  TIP60 complex is a kind of HAT ◦ HAT : Histone AcetylTransferase  TIP60 complex acetylates H4 and  H2AX  AcH4 is very critical factor for DDR ◦ Mutation -> Hypersensitive

21.  Tip49a,b(also known as Rvb1, 2) are involved to TRRAP/Tip60 activity(HAT activity)  Knockdown of Rvb1,2 -> increase of  H2AX Jha S, Mol Cell Biol 2008; 28:2690-700.

22.  Why?  AcH4 is important histone to recruit phosphatase  Chromatin Remodeling Factor binds to N-ter of AcH4 and remove  H2AX

23.  TRRAP/Tip60 relax histone to recruit DNA repair protein effectvely.  Mutation of these proteins -> Reduce recruitment of 53BP1 and Rad51 -> permit to access DNA to open up

24.  Mono or poly ubiquitin can be signal factor

25.  UBC13 can interact RNF8 and RNF168 and TIP60 ◦ RNF8 : E3 ligase ◦ RNF168 : E3 ligase ◦ UBC13 : E2 ligase  Nature Reviews Cancer 6, 369-381 (May 2006)  doi:10.1038/nrc1881

26.  Binds to phosphorylated MDC1 by FHA domain  FHA domain : forkhead associated domain  Ubiquitynate on H2AK63 or H2AXK63  RAP80 can bind to these histones  Abraxas(adaptor protein) binds to RAP80  BRCA1 binds to Abraxas 

27.  Binds to monoUbH2AX K63  With UBC13, makes polyUbH2AX K63

28.  TIP60 forms a complex with UBC13 to promote the DNA damage dependent ubiquitylation of Ac  H2AX  Acetylated site is for its ubiquitylation  UBC13 and TIP60 complex is related to histone removal

31.  H3K79 and H4K20 are important residues  They are methylated in normal, and exposed at DSB  They are docking sites for recruting signal transducer

32.  53BP1 contains two tandem TUDOR domain  TUDOR domain can bind methylated H3 and H4

33.  Williams Beuren syndrome factor transcription factor -> Tyrosine kinase  -> phosphorylated T142 of H2AX