lumir krejci lord, laboratory of recombination and dna repair national centre for biomolecular...
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Lumir Krejci
LORD, Laboratory of Recombination and DNA Repair
National Centre for Biomolecular Research& Department of Biology
DNA Repair and Genomic Instability
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Jaký mechanismus se podílí na opravě
dvouřetězcových zlomů?
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Why do we study this?
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Common Types of DNA Damage and Spontaneous Alterations
RadiotherapyIonizing RadiationX-rays
Chemotherapy(Alkylating agents)Cisplatin Mitomycin CCyclophosphamidePsoralenMelphalan
UV (sunlight)Pollution (hydrocarbons)
Exogenous Sources
Endogenous SourcesOxidative damage by free radicals (oxygen metabolism)Replicative errorsSpontaneous alterations in DNAAlkylating agents (malondialdehyde)
SmokingFoodstuffs
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• loss of base – 26,000
• deamination of cytosin – 1 000
• alkylation of base – x 10 000
• dimerization of pyrimidins – 50 000
• ssDNA breaks – 100,000
Total ~ 500 000 damage/day
DNA damage in human cell per day:
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DNADamageMetabolismMetabolism
Exogenous
Endogenous
DNAReplication
Permanent Genetic
Alteration
Disease
CellCycleArrest
DNARepair
Apoptosis
Failure to repair DNA damage:
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1. Direct reversals2. Excision repair
- Base Excision Repair (BER) - Nucleotide Excision Repair (NER)
3. Mismatch repair (MMR) - replication errors
4. Recombinational repair (HR and NHEJ) - multiple pathways - double strand breaks and interstrand cross-links
5. Tolerance mechanisms - lesion bypass (TLS) - recombination
DNA Repair Pathways
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Damage Recognized:Thymine dimers6-4 photoproduct
Gene Products Required:Photolyase
Related disease:Photolyase not yet found in placental mammals
T T
Visible light
T T
Repair by Direct reversal:
photoreactivation
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Damage Recognized:
- Base deamination- Oxidative damage- and other minor base modifications
Gene Products Required (5):
- Glycosylase- AP endonuclease- Phosphodiesterase- DNA polymerase- DNA Ligase
E. coli Base Excision Repair (BER)
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XPB & XPD - DNA helicases
XPC - damage recognition
XPA & RPA - damage validation & complexstabilization
XPG - 3’ incision
ERCC1-XPF - 5’ incision
(junction specific endonucleases)
CSA & CSB - role in processing RNAP II?XPC not required
Replication-coupled NERReplication-coupled NER
Nucleotide Excision Repair
E. coli 5’ incision is 8 nuc. from lesion 3’ incision is 4 nuc. from lesion
Mammals 5’ incision is 22 nuc. from lesion 3’ incision is 6 nuc. from lesion
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Xeroderma Pigmentosum (classical)• Occurrence: 1-4 per million population• Sensitivity: ultraviolet radiation (sunlight)• Disorder: multiple skin disorders; malignancies of the
skin; neurological and ocular abnormalities• Biochemical: defect in early step of NER• Genetic: autosomal recessive, seven genes (A-G)
Xeroderma Pigmentosum (variant)• Occurrence: same as classical• Sensitivity: same as classical• Disorder: same as classical• Biochemical: defect in translesion bypass
Genetics of NER in Humans
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Cockayne’s Syndrome• Occurrence: 1 per million population• Sensitivity: ultraviolet radiation (sunlight)• Disorder: arrested development, mental retardation,
dwarfism, deafness, optic atrophy, intracranial calcifications; (no increased risk of cancer)
• Biochemical: defect in NER• Genetic: autosomal recessive, five genes (A, B and XPB, D & G)
Genetics of NER in Humans
Trichothiodystrophy• Occurrence: 1-2 per million population• Sensitivity: ultraviolet radiation (sunlight)• Disorder: sulfur deficient brittle hair, mental and growth
retardation, peculiar face with receding chin, ichthyosis;(no increased cancer risk)
• Biochemical: defect in NER• Genetic: autosomal recessive, three genes (TTDA,
XPB, XPD)
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Repair of Replication Errors
Mechanisms for Insuring Replicative Fidelity
1. Base pairing 10-1 to 10-2
2. DNA polymerases 10-5 to 10-6
- base selection- proofreading
3. Accessory proteins 10-7
- single strand binding protein4. Mismatch correction 10-10
Further reading: A. Bellacosa, Cell Death and Differentiation 8, 1076 (2001) M. J. Schofield & P. Hsieh, Ann. Rev. Microbiol. 57, 579 (2003)
DNA Mismatch Repair
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Mismatch Repair
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Mismatch Repair Mutations inHereditary Nonpolyposis Colon
Cancer (HNPCC)
• MMR mutations in 70% of families
• MLH1 (50%), MSH2 (40%)
• Minor role for MSH6, PMS1, PMS2
• Population prevalence 1:2851 (15-74 years)
• 18% of colorectal cancers under 45 years
• 28% of colorectal cancers under 30 years
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Lesions repaired1. Double-strand breaks2. Interstrand cross-links
Further reading: Paques and Haber, Microbiol. & Molec. Biol. Rev. 63, 349 (1999)
Recombinational DNA Repair Mechanisms
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Translesion Bypass DNA Polymerases
Pol eta- inserts adenosines opposite TT dimers- in general has low fidelity- low processivity- may be error-prone with other lesions- Pol eta is a product of the XPV gene
Pol zeta and Rev 1- Rev 1 inserts random bases opposite dimer- Pol zeta extends bypass by a few bases- Both polymerases have low fidelity and low processivity
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Cross-link repairModel for the mechanism of
DNA ICL repair in mammalian cells.
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Richard D. Kennedy et al. Genes Dev. 2005; 19: 2925-2940
Schematic interaction of the FA pathway
L=catalytic element?
=BRIP1
and BRCA1, RAD51, PCNA, NBS1
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Fanconi’s Anemia
Congenital abnormalities - skeletal - skin pigmentation - short stature - male genital - mental retardation - cardiac abnormalities - hearingCancer - myeloid leukemia - solid tumors
Review: Tischkowitz & Hodgson, J. Med. Genet. 40, 1 (2003)
13 genes in FABRCA2 is deficient in FA-D1
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Cross-link repair
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What do we study?
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• Induced by ionizing radiation & chemicals
• Arise when replicating a damaged template
• Serve as the initiator of meiotic recombination
• Part of immune response
DNA double-strand breaks (DSB)
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• Cell death
• Chromosomal aberrations
• Meiotic aneuploidy
• Immunodeficiency
Failure to properly process DSBs
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Adapted from Surralles et al., Genes Dev. (2004)
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
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End processing
SAE2
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Homology search
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PolRAD54
DNA repair synthesis
SRS2
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SRS2MUS81
MMS4
Resolution
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Ligation
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How do we study this?
Examples - Regulation of recombination?
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Presynaptic Rad51 filament
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Positive regulation - Mediator proteins
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Proteins
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DNDNA bindingA binding
Seong et al. J. Biol. Chem., 2008
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Function of Rad52 protein
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• Can interfere with normal repair
• Elicits strong cell cycle responses
• Causes cell death
Negative regulation - Recombination can be
harmfull to cells:
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Cells have ways to prevent untimely recombination!
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Srs2 binds Rad51
(Krejci et al, Nature 2003)
Krejci et al. NATURE, 2003
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EM of Rad51 filaments
(Krejci et al, Nature 2003)Krejci et al. NATURE, 2003
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Protein modification
by SUMOylation
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Rad52 is SUMOylated
Co to je sumoylace
V. Altmannova
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Quality control mechanism Quality control mechanism
Rad52
Rad51 Srs2
RPA
RPA