DNA damage response DNA damage response and chromatin remodelingand chromatin remodeling

Presentation for Ron Shamir group internal meetingPresentation for Ron Shamir group internal meeting

3/11/20103/11/2010

DNA damages, including single- and double- strand DNA damages, including single- and double- strand

breaks (breaks (SSBSSB and and DSBDSB respectively), pyrimidine respectively), pyrimidine

dimmers (dimmers (PDPD), and oxidized nucleotides, are caused ), and oxidized nucleotides, are caused

by physical agents in the environment, like ionizing by physical agents in the environment, like ionizing

radiation (radiation (IRIR) and ultra violet light () and ultra violet light (UVUV), and by ), and by

various chemicals (including reactive oxygen various chemicals (including reactive oxygen

species (species (ROSROS) from internal metabolic processes ) from internal metabolic processes

source). source).

In eukaryotes, the sensing and repair of DNA In eukaryotes, the sensing and repair of DNA

damages occur in chromatin context. damages occur in chromatin context. Chromatin Chromatin

constitutes a physical barrier to enzymes and constitutes a physical barrier to enzymes and

regulatory factors to reach the DNA.regulatory factors to reach the DNA. To deal with this To deal with this

obstacle, transient chromatin structural changes are obstacle, transient chromatin structural changes are

integral to base- and nucleotide-excision, homologous integral to base- and nucleotide-excision, homologous

recombination, and non-homologous end joining DNA recombination, and non-homologous end joining DNA

repair pathways. repair pathways.

DDR and Chromatin RemodelingDDR and Chromatin Remodeling

Nucleotide Excision Repair (NER)Nucleotide Excision Repair (NER)

Nucleotide Excision Repair (NER)

Base Excision Repair (BER)Base Excision Repair (BER)

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

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.

Histone-histone associations

The four core histones tend to associate with each other in very specificpatterns of interactions. Histones H3 and H4 interact very strongly with each otherand form a specific complex, a tetramer. Histones H2A and H2B alsointeract with each other and can form primarily dimers and some higher oligomers.

The pattern of association is:

H3 H4

H2A H2B

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.

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

nm Chromatinnm Chromatin

Chromatin: H1 histoneChromatin: H1 histone

H1 histone just helps to tightened the nucleosome

The Organization of ChromatinThe Organization of Chromatin

Hansen, J.C., Annu Rev Biophys Biomol Struct. 2002

Chromatin: levelsChromatin: levels of condensation of condensation

Deposition of histones during Deposition of histones during replicationreplication

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

Chromatin remodeling mechanisms includes:Chromatin remodeling mechanisms includes:

(1)(1) posttranslational modifications (PTMs) of histones and posttranslational modifications (PTMs) of histones and

non-histones chromatin associated proteins, including non-histones chromatin associated proteins, including

covalent additions (removals) ofcovalent additions (removals) of phosphoryl-, methyl-, phosphoryl-, methyl-,

acetyl-, ubiquitin-, SUMO, and ADP-ribose acetyl-, ubiquitin-, SUMO, and ADP-ribose

(2) histone-variants replacement, and nucleosomes (2) histone-variants replacement, and nucleosomes

repositioning or eviction. ATP-dependent multi subunits repositioning or eviction. ATP-dependent multi subunits

chromatin remodeling complexeschromatin remodeling complexes (INO80, SRCAP/SWR-(INO80, SRCAP/SWR-

C, SWI/SNF, and MI-2/NuRD) implement the latter C, SWI/SNF, and MI-2/NuRD) implement the latter

mechanisms mechanisms

Enzymes that regulate chromatinEnzymes that regulate chromatin

Two classes of enzymes that regulate Two classes of enzymes that regulate chromatin structure: chromatin structure: histone modifiershistone modifiers and and chromatin remodelerschromatin remodelers..

Histone modifiers don’t alter nucleosome Histone modifiers don’t alter nucleosome positionposition; they make passive marks that recruit ; they make passive marks that recruit more active functions (histone code).more active functions (histone code).

Chromatin remodelers Chromatin remodelers hydrolyze ATPhydrolyze ATP to to actively remodel chromatin: shift nucleosome actively remodel chromatin: shift nucleosome position with respect to DNA, exposing or position with respect to DNA, exposing or occluding regulatory sequences.occluding regulatory sequences.

These enzymes function within larger These enzymes function within larger complexes of subunits that collectively act to complexes of subunits that collectively act to enhance and/or target the remodeling activity.enhance and/or target the remodeling activity.

ATP-dependant chromatin remodeling ATP-dependant chromatin remodeling complexescomplexes

SWI/SNF:: 15 subunits complex. Catalytic subunits are 15 subunits complex. Catalytic subunits are SMARCA2 or SMARCA4. 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 subunits complex. incorporate the histone variant H2AFZ into nucleosomes.histone variant H2AFZ into nucleosomes.

INO80: 16-subunits complex. Involved in 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 13 subunits complex. The Catalytic subunits are CHD3 and CHD4. Involved in the regulation of some important CHD3 and CHD4. Involved in the regulation of some important DDR genes like P53, BRCA1 and MCPH1 .DDR genes like P53, BRCA1 and MCPH1 .

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

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 facilitate

DNA repair beyond their well-documented role in DNA repair beyond their well-documented role in

transcription, presumably by opening or loosening transcription, presumably by opening or loosening

compact nucleosomal structure close to sites of damage.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

Nucleosome tail Nucleosome tail modificationsmodifications

Lysine acetylations.Lysine acetylations.• Histone Acetyl-Histone Acetyl-

Transferases (HAT) & Transferases (HAT) & Histone Deacetylases Histone Deacetylases (HDAC).(HDAC).

Lysine and Arginine Lysine and Arginine Metylations.Metylations.

Modified by histone-Modified by histone-metyl-transferase.metyl-transferase.

Phosphorilation.Phosphorilation. Ubiquitination.Ubiquitination.

H2A ubiquitination affects H2A ubiquitination affects 10-15% of this histone in 10-15% of this histone in most eukaryotic cells most eukaryotic cells

ADP-ribosylation. ADP-ribosylation.

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.

Chromatin remodeling Chromatin remodeling is an is an activeactive process process

Chromatin remodeling describes the energy-Chromatin remodeling describes the energy-dependent (ATP) displacement or reorganization dependent (ATP) displacement or reorganization of nucleosomes that occurs in conjunction with of nucleosomes that occurs in conjunction with activation (activation (or repressionor repression) of genes for ) of genes for transcriptiontranscription

Chromatin remodelers also play a role in Chromatin remodelers also play a role in recombination and repairrecombination and repair

Chromatin Remodeling ComplexesChromatin Remodeling Complexes

PARP1PARP1 and theand the DDB1-2-CUL4ADDB1-2-CUL4A complex are early complex are early

sensors of SSB, DSB, and PD DNA lesions. These sensors of SSB, DSB, and PD DNA lesions. These

proteins have chromatin-remodeling enzymatic proteins have chromatin-remodeling enzymatic

activity. Additional early DSB sensors are the activity. Additional early DSB sensors are the MRN MRN

and and Ku70-Ku80Ku70-Ku80 complexes, that activate complexes, that activate ATMATM andand

DNA-PKcs DNA-PKcs kinases respectively, both belonging to the kinases respectively, both belonging to the

phosphoinositide-3-kinase-related protein kinases phosphoinositide-3-kinase-related protein kinases

(PIKK).(PIKK). Upon SSB repair pathways, Upon SSB repair pathways, ATRATR, a third , a third

member of the PIKK kinases is activated. member of the PIKK kinases is activated.

The The PIKKPIKK kinases phosphorylate many substrates, kinases phosphorylate many substrates,

including histones and proteins involved in regulation including histones and proteins involved in regulation

of chromatin structure. One of these proteins is of chromatin structure. One of these proteins is

TRIM28 (KAP1),TRIM28 (KAP1), a transcription co-repressor a transcription co-repressor

proposed to regulate chromatin structure and proposed to regulate chromatin structure and

heterochromatin formation. In response to the heterochromatin formation. In response to the

induction of DNA DSBs, its co-repression function is induction of DNA DSBs, its co-repression function is

inhibited by ATM-dependant phospohorylation. inhibited by ATM-dependant phospohorylation.

The main histone PTMs in response to DSBs The main histone PTMs in response to DSBs

are those related to are those related to H2AFXH2AFX (histone (histone H2A H2A

variant). DDR-related replacement of H2A variant). DDR-related replacement of H2A

variant in the nucleosomes by variant in the nucleosomes by H2AFZ H2AFZ

(promoted by the (promoted by the SRCAP/SWR-C ATP-SRCAP/SWR-C ATP-

dependent complexdependent complex) also occur. ) also occur.

Upon DSB, the histone acetylase complex Upon DSB, the histone acetylase complex

NUA4/TIP60NUA4/TIP60, several histones ubiquitin ligases , several histones ubiquitin ligases

are activated. The interactions between the are activated. The interactions between the

DDR proteins, DDR proteins, MCPH1MCPH1, , P53P53 and and BRCA1BRCA1, and , and

components of the components of the SWI/SNF SWI/SNF and and MI-2/NuRD MI-2/NuRD

chromatin remodeling complexes, seems to be chromatin remodeling complexes, seems to be

important to the regulation of DDR. important to the regulation of DDR.

In conclusion:In conclusion:In eukaryotic cells, packaging of DNA into highly In eukaryotic cells, packaging of DNA into highly condensed chromatin presents a significant obstacle to condensed chromatin presents a significant obstacle to DNA-based processes. (replication, repair and DNA-based processes. (replication, repair and recombination).recombination).

Cells use three major strategies to allows protein factors Cells use three major strategies to allows protein factors to gain access to nucleosomal DNA:to gain access to nucleosomal DNA:

1. Histone posttranslational modifications (PTMs)

2. Incorporation of histone variants into nucleosomes

3. ATP-dependent chromatin remodeling

ATP-dependent multi subunits chromatin ATP-dependent multi subunits chromatin

remodeling complexesremodeling complexes (INO80, SRCAP/SWR- (INO80, SRCAP/SWR-

C, SWI/SNF, and MI-2/NuRD)C, SWI/SNF, and MI-2/NuRD) implement the implement the

latter mechanisms and many proteins that are latter mechanisms and many proteins that are

involved in DNA repair and DDR have physical involved in DNA repair and DDR have physical

interactions and reciprocal regulations with interactions and reciprocal regulations with

some proteins that belongs these complexes.some proteins that belongs these complexes.