DNA damage response and chromatin remodeling Presentation for Ron Shamir group internal meeting

1. DNA damage response and chromatin remodeling Presentation for Ron Shamir group internal meeting 3/11/2010

2. DNA damages, including single- and double- strand breaks (SSB and DSB respectively), pyrimidine dimmers (PD), and oxidized nucleotides, are caused by physical agents in the environment, like ionizing radiation (IR) and ultra violet light (UV), and by various chemicals (including reactive oxygen species (ROS) from internal metabolic processes source).

3. In eukaryotes, the sensing and repair of DNA damages occur in chromatin context. Chromatin constitutes a physical barrier to enzymes and regulatory factors to reach the DNA. To deal with this obstacle, transient chromatin structural changes are integral to base- and nucleotide-excision, homologous recombination, and non-homologous end joining DNA repair pathways.

4. DDR and Chromatin Remodeling

6. Nucleotide Excision Repair (NER) • Nucleotide Excision Repair (NER)

7. Base Excision Repair (BER)

8. HDAC (Histone Deacetylase) Families • Class I and II: • Zinc-hydrolase • ~11 human homologs • Inhibited by hydroxamic acid containing compounds (TSA, SAHA) • Class III (Sir2 or sirtuins) • No structural/mechanistic similarity to Class I and II • NAD+ requiring • 7 human homologs • Inhibited by nicotinamide Histone modification and DDR

9. The basic structural subunits of the chromatin are thenucleosomes. These core particles consists of about 146 bp of dsDNA wrapped in 1.65 left-handed superhelical turns around four identical pairs of the histone proteins H2A, H2B, H3 and H4, collectively known as the histone octamer. The remaining 50 bp of the repeating unit consists of "linker DNA", dsDNA which separates the core particles.

11. Histone-histone associations The four core histones tend to associate with each other in very specific patterns of interactions. Histones H3 and H4 interact very strongly with each other and form a specific complex, a tetramer. Histones H2A and H2B also interact with each other and can form primarily dimers and some higher oligomers. The pattern of association is: H3 H4 H2A H2B

13. The crystal structure of the nucleosome core particle consisting of histone proteins H2A , H2B , H3 and H4. (Luger & Richmond) Nature. 1997 Sep 18; 389 (6648): 251-60.

14. N.4. Chromatin: A Speculative Model for the Formation of 30 nm Chromatin

15. Chromatin: H1 histone H1 histone just helps to tightened the nucleosome

17. The Organization of Chromatin Hansen, J.C., Annu Rev Biophys Biomol Struct. 2002

18. Chromatin: levelsof condensation

19. Deposition of histones during replication During replication, parental histones are distributed randomly onto the replicated DNA strands, and newly synthesized histones fill the gaps. http://www.umassmed.edu/faculty/show.cfm?start=0&faculty=912

20. Chromatin remodeling mechanisms includes: • (1)posttranslational modifications (PTMs) of histones and non-histones chromatin associated proteins, including covalent additions (removals) ofphosphoryl-, methyl-, acetyl-, ubiquitin-, SUMO, and ADP-ribose • (2) histone-variants replacement, and nucleosomes repositioning or eviction. ATP-dependent multi subunits chromatin remodeling complexes(INO80, SRCAP/SWR-C, SWI/SNF, and MI-2/NuRD) implement the latter mechanisms

21. Enzymes that regulate chromatin • Two classes of enzymes that regulate chromatin structure: histone modifiersand chromatin remodelers. • Histone modifiers don’t alter nucleosome position; they make passive marks that recruit more active functions (histone code). • Chromatin remodelers hydrolyze ATP to actively remodel chromatin: shift nucleosome position with respect to DNA, exposing or occluding regulatory sequences. • These enzymes function within larger complexes of subunits that collectively act to enhance and/or target the remodeling activity.

22. ATP-dependant chromatin remodeling complexes • SWI/SNF:15 subunits complex. Catalytic subunits are SMARCA2 or SMARCA4. plays essential roles in a variety of cellular processes including differentiation, proliferation and DNA repair. • SRCAP/SWR-C:10 subunits complex. incorporate the histone variant H2AFZ into nucleosomes. • INO80:16-subunits complex. Involved in DNA repair, checkpoint regulation, and DNA replication, cooperatively with their histone substrates,gamma-H2AX and H2AFZ. • Mi-2/NuRD:13 subunits complex. The Catalytic subunits are CHD3 and CHD4. Involved in the regulation of some important DDR genes like P53, BRCA1 and MCPH1 .

23. Histone PTMs influence genome function by: • directly disturbing nucleosome stability which affects chromatin compaction and accessibility • constituting a docking site for different kinds of non-histone proteins • Certain histone PTMs indicate the specific position of DNA damage and provide a platform of interaction for DDR proteins, determining the repair pathway that should be involved

24. The best-known histone PTM is histone acetylation - deacetylation, controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Histone acetylations enhance chromatin accessibility. and facilitate DNA repair beyond their well-documented role in transcription, presumably by opening or loosening compact nucleosomal structure close to sites of damage. • Histone phosphorylation mediated by members of the PI3K kinase superfamily (ATM, ATR, DNA-PK) plays a role at the beginning of DDR by facilitating the access of different repair proteins to DNA breaks

25. Nucleosome tail modifications Lysine acetylations. Histone Acetyl-Transferases (HAT) & Histone Deacetylases (HDAC). Lysine and Arginine Metylations. Modified by histone-metyl-transferase. Phosphorilation. Ubiquitination. H2A ubiquitination affects 10-15% of this histone in most eukaryotic cells ADP-ribosylation.

26. Acetylation of Lysine 16 of histone H4 completely abolishes the ability of the tail domains to mediate nucleosome-nucleosome interactions, which are required for chromatin condensation.

27. Chromatin remodeling is an active process • Chromatin remodeling describes the energy-dependent (ATP) displacement or reorganization of nucleosomes that occurs in conjunction with activation (or repression) of genes for transcription • Chromatin remodelers also play a role in recombination and repair

28. Chromatin Remodeling Complexes

29. PARP1and theDDB1-2-CUL4A complex are early sensors of SSB, DSB, and PD DNA lesions. These proteins have chromatin-remodeling enzymatic activity. Additional early DSB sensors are the MRN and Ku70-Ku80 complexes, that activate ATMandDNA-PKcs kinases respectively, both belonging to the phosphoinositide-3-kinase-related protein kinases (PIKK). Upon SSB repair pathways, ATR, a third member of the PIKK kinases is activated.

31. The PIKK kinases phosphorylate many substrates, including histones and proteins involved in regulation of chromatin structure. One of these proteins is TRIM28 (KAP1), a transcription co-repressor proposed to regulate chromatin structure and heterochromatin formation. In response to the induction of DNA DSBs, its co-repression function is inhibited by ATM-dependant phospohorylation.

32. The main histone PTMs in response to DSBs are those related to H2AFX (histone H2A variant). DDR-related replacement of H2A variant in the nucleosomes by H2AFZ (promoted by the SRCAP/SWR-C ATP-dependent complex) also occur.

34. Upon DSB, the histone acetylase complex NUA4/TIP60, several histones ubiquitin ligases are activated. The interactions between the DDR proteins, MCPH1, P53 and BRCA1, and components of the SWI/SNF and MI-2/NuRD chromatin remodeling complexes, seems to be important to the regulation of DDR.

35. In conclusion: In eukaryotic cells, packaging of DNA into highly condensed chromatin presents a significant obstacle to DNA-based processes. (replication, repair and recombination). Cells use three major strategies to allows protein factors to gain access to nucleosomal DNA: 1. Histone posttranslational modifications (PTMs) 2. Incorporation of histone variants into nucleosomes 3. ATP-dependent chromatin remodeling

36. ATP-dependent multi subunits chromatin remodeling complexes (INO80, SRCAP/SWR-C, SWI/SNF, and MI-2/NuRD)implement the latter mechanisms and many proteins that are involved in DNA repair and DDR have physical interactions and reciprocal regulations with some proteins that belongs these complexes.