DNA damage response

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

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• 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).

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• 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.

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DDR and Chromatin Remodeling
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• Nucleotide Excision Repair (NER)

Nucleotide Excision Repair (NER)
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• Base Excision Repair (BER)

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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
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• The basic structural subunits of the chromatin
  are the nucleosomes. 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.

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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
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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.
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N.4. Chromatin A Speculative Model for the
Formation of 30 nm Chromatin
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Chromatin H1 histone
H1 histone just helps to tightened the nucleosome
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The Organization of Chromatin
Hansen, J.C., Annu Rev Biophys Biomol Struct. 2002
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Chromatin levels of condensation
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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?start0f
aculty912
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• Chromatin remodeling mechanisms includes
• (1) posttranslational modifications (PTMs) of
  histones and non-histones chromatin associated
  proteins, including covalent additions (removals)
  of phosphoryl-, 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

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Enzymes that regulate chromatin

• Two classes of enzymes that regulate chromatin
  structure histone modifiers and chromatin
  remodelers.
• Histone modifiers dont 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.

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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 .

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• 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

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• 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

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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.

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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.
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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

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Chromatin Remodeling Complexes
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• PARP1 and the DDB1-2-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 ATM
  and DNA-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.

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• 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.

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• 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.

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• 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.

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• 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

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• 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.