Repair of DNA and XP Errol C. Friedberg (2003) DNA damage and repair. Nature 421: 436-440.

Repair of DNA and XP

Errol C. Friedberg (2003) DNA damage and repair.

Nature 421: 436-440.

Xeroderma Pigmentosum (XP)

Symptoms include:

---Extreme sensitivity to sunlight

---Early onset of skincancer

NER

HealthyDNA

Why are XP patients sensitive to sunlight?

HealthyDNA

UV

DamagedDNA

NO REPAIR

8 gens

Xeroderma Pigmentosum (XP)

Hereditary Cancer Syndromes involving defects in DNA Repair

• Xeroderma pigmentosum..........Nucleotide Excision Repair

• Hereditary Nonpolyposis......... Colon Cancer Mismatch DNA Repair

• Ataxia Telangiectasia............... Double-Strand Break

• Fanconi Anemia DNA ..............Crosslink Repair

• Li-Fraumeni ..............................Nucleotide Excision Repair

• Breast-Ovarian Cancer.............. Double Strand Breaks

Nucleotide Excision Repair

DNADamageMetabolism

Exogenous

Endogenous

DNAReplication

Permanent Genetic

Alteration

Disease

CellCycleArrest

DNARepair

Apoptosis

Daño y reparación del DNA

• Base Loss• Base modification & Deamination

• Chemical Modification• Photodamage• Inter-strand crosslinks

• DNA-protein crosslinks• Strand breakage

Types of DNA damage

Abasic site -loss of a nucleobase(apurinic or apyrimidinic)

Deamination

Base loss

Potential Sites of modification/damage

Chemical Damage

Oxidative damage

Alkylation

UV-induced damage

Pyrimidine dimers

Note: Cytosine residues can also form dimers

Types of Damage Repair

• Photolyase• De-alkylation proteins (not catalytic)

• Base Excision Repair• Nucleotide Excision Repair (GG and TC)

• Recombination Repair• Error-prone Repair• Double strand Break Repair (if time permits)

UV-responsive photolyases

Direct reversal (de-alkylating proteins)

Base Excision Repair

Base Excision Repair

BER

NER

Nucleotide Excision Repair

(E.coli)

Nucleotide Excision Repair (Global Genome Repair -Humans)

Nucleotide Excision Repair(Transcription Coupled -Humans)

NER

Common features of GGR & TCR

Mismatch Repair

Recombination Repair

Other possible mechanisms of recombination repair

Mecanismos de Reparación del DNA (DSB)

Recombinacion Homologa

RAD50

MRE11

NSB1

Error Prone Bypass (E. coli)

Experimental evidence for Error prone repair(E.coli)

Revertant in His- genes(umuC mutated strain)

UV-responsive activation of the umuC gene

DNA repair polymerases

DNA polymerase Eta (XP-V) - addition of two dA residues across pyrimidine dimers

DNA polymerase Zeta - addition of random residues across pyrimidine dimers

Non-Homologous End Joining (Double Strand Breaks)

Model for activation of DNA damage repair

Damage & Repair

• Multiple forms of DNA damage occur• These are repaired constantly by several

mechanisms • Failure to repair damage leads to mutations• Often defects in damage sensing machinery

or DNA repair processes can be correlated with increased incidence of diseases such as cancer

Factors involved in Damage Sensing

Apoptosis and DNA RepairApoptosis and DNA Repair

• Objectives:– 1) Understand that programmed cell death or apoptosis is genetically controlled and is an important factor in tumour growth

– 2) Describe the relationship of abnormal DNA repair to genetic instability and cancer-prone syndromes

– 3) Describe DNA-damage activated cell-cycle checkpoints and how they prevent mutation and abnormal cellular division

Van Gent et al, 2001

The The ImportanceImportanceof of DNA-dsbDNA-dsbRepairRepair

Van Gent et al, 2001

Molecular DNA-dsb Repair

BRCA1/2

DNA Damage-Induced DNA Damage-Induced Protein InteractionsProtein Interactions

Nbs1 Mre11 DAPI Merged

Time after IR (hrs)

0 0.5 3 6 12

H2AXBP53BRCA1 Rad50, Rad51

Indicators or Biomarkers of DNA Repair ?

BRCA1/BRCA2BRCA1/BRCA2• Tumour suppressor genes associated with high risk of

breast cancer at young age with bilateral tumour risk (BRCA1 also ovarian and prostate/colon; BRCA2 also ovarian, male breast)

• These mutations account for 80% of familial breast cancer; yet only 5-10% of breast cancers are familial, the others are sporadic

• NOT associated with sporadic breast cancer (unlike p53)• Both proteins involved in the repair of DNA double strand

breaks and predispose to aberrant DNA replication and lead to mutations and cancer

ATM

BRCA1/2 proteins may have a role in homologous DNA-BRCA1/2 proteins may have a role in homologous DNA-dsb repair due to ATM, rad50 and rad51 interactions and dsb repair due to ATM, rad50 and rad51 interactions and mutant BRCA1/2 proteins can not interact appropriatelymutant BRCA1/2 proteins can not interact appropriately

HNPPC and Mismatch RepairHNPPC and Mismatch Repair

• Hereditary non-polyposis colorectal cancer (HNPPC) is a disorder of faulty mismatch repair and genetic instability

• evolution of multiple tumours occurs much more rapidly at young age (<50 yrs) and accounts for 2-4% of all colonic cancers

• Have decreased ability to repair replication errors (RER+ phenotype) due to a lack of the MLH1 or MSH2 genes which normally remove incorrect DNA base pairs

• Leads to DNA microsatellite instability (MSI) detectable on gel electrophoresis; interestingly MSI+ colon cancers have a better prognosis following therapy

Microsatellite Instability (tandom repeats of DNA) isMicrosatellite Instability (tandom repeats of DNA) isa sign of MMR-deficiency and may be due to MSH2-MLH1a sign of MMR-deficiency and may be due to MSH2-MLH1

mutations (IHC) = Diagnostic Testingmutations (IHC) = Diagnostic Testing

Chromosomal Control and Chromosomal Control and Cancer: TelomeresCancer: Telomeres

• Cells are capable of only a limited series of divisions before they arrest or senesce and chromosomal fusions and cell death

• Telomeres caps of chromosome ends and function to prevent DNA loss during DNA replication and provide a cellular clock for cell proliferation

• They consist of a specific sequence TTAGGGG associated with proteins (TEP, hTERT)

• As somatic cells normally age, telomeres reduce in length due to decreased function of telomerase, an enzyme which is used by germline cells to maintain telomere length = Cancer Preventionor Tumour Suppression

• In human tumours, telomerase activity is abnormally high leading to abnormal control of cell growth and proliferation

Normal and Abnormal Telomere FunctioningNormal and Abnormal Telomere FunctioningIn Normal or Cancer CellsIn Normal or Cancer Cells

GTTAGGGTTA

5'3'

TEP1hTERT

TEMPLATE

INACTIVE ACTIVE

shortened telomeres

cell senescence

stable telomeres

cell survival

AGING CANCER

TELOMERASE

Harrington, 2001

SUMMARYSUMMARY-Apoptosis is triggered by a number of external stressors including chemo- or radiotherapy and is controlled by the p53, myc and bax-bcl-2 proteins-Distinct morphologic features define apoptotic cells secondary to caspase activity -The relative level of apoptosis versus cell proliferation determines selection of those mutant cells which may proliferate to form a tumour

-Defects in DNA repair of UV-damage, DNA replication errors orDNA-dsbs can lead to genetic instability and genetic mutation -These defects lead to cancer-prone syndromes such as xerodermapigmentosum, HNPPC and BRCA1-associated tumours in whichpatients are sensitive to specific DNA-damaging agents, develop cancers at an early age and have chromosomal instability in tissues