Jan H.J. Hoeijmakers, Ph.D. MOLECULAR ORIGINS OF CANCER DNA Damage, Aging, and Cancer Student: Alia...

Jan H.J. Hoeijmakers, Ph.D.

MOLECULAR ORIGINS OF CANCER DNA Damage, Aging ,

and Cancer

Student: Alia Alqneibi Bch 550 2nd semester 1341

Supervisor/Dr Gihan Gawish

FirstFirst spontaneous reactions (mostly spontaneous reactions (mostly hydrolysis) intrinsic to the chemical hydrolysis) intrinsic to the chemical nature of DNA in an aqueous solution nature of DNA in an aqueous solution create a basic sites and cause create a basic sites and cause

deaminationdeamination..

SecondSecond our own metabolism our own metabolism generates:generates:

-Reactive oxygen and nitrogen species. -Reactive oxygen and nitrogen species. - lipid peroxidation products. - lipid peroxidation products. - Endogenous alkylating agents.- Endogenous alkylating agents. -Estrogen and cholesterol metabolites. -Estrogen and cholesterol metabolites. -And reactive carbonyl species.-And reactive carbonyl species.

14 all of which damage DNA.14 all of which damage DNA.

DNA integrity is threatened fromDNA integrity is threatened from three sidesthree sides

ThirdThird DNA is damaged by exogenous physical and chemical DNA is damaged by exogenous physical and chemical agents, but this damage is to some extent avoidable.agents, but this damage is to some extent avoidable.

ContCont..

DNA injury can induce mutations that cause cancer or cell DNA injury can induce mutations that cause cancer or cell death or senescence, contributing to aging.death or senescence, contributing to aging.

The type of damage that occurs is important for the type of The type of damage that occurs is important for the type of outcome.outcome.

Some lesions are primarily mutagenic, others mainly cytotoxic Some lesions are primarily mutagenic, others mainly cytotoxic or cytostatic.or cytostatic.

Many DNA lesions lead to both types of outcomes in different Many DNA lesions lead to both types of outcomes in different ratios, depending on:ratios, depending on:

-The location and number of lesions, -The location and number of lesions, -Cell type, -Cell type,

-And stage in the cell cycle and differentiation-And stage in the cell cycle and differentiation..

DNA injuryDNA injury

Consequences of DNA Consequences of DNA damagedamage

LONG-TERM CONSEQUENCESLONG-TERM CONSEQUENCES

Ageing Cancer Disease

DNA REPAIR MECHANISMSDNA REPAIR MECHANISMS

SHORT-TERM CONSEQUENCESSHORT-TERM CONSEQUENCES

ABNORMAL GROWTH & METABOLISM

PHYSIOLOGICAL DYSFUNCTION

CELL DEATH

Decreased cellular proliferation

Defective signaling pathways Impaired protein/ gene expression

Genomic instability

DNA DAMAGE

The genomic maintenance system includes at The genomic maintenance system includes at least six multistep repair pathways, each least six multistep repair pathways, each covering a specific subclass of DNA lesion.covering a specific subclass of DNA lesion.

The genomic maintenanceThe genomic maintenancesystemssystems

A-A- Double-strand breaks (DSBs) in DNA are highly Double-strand breaks (DSBs) in DNA are highly cytotoxic and cytostatic forms of damage.cytotoxic and cytostatic forms of damage.

They are repaired through:They are repaired through: 1- Nonhomologous end-joining (NHEJ).1- Nonhomologous end-joining (NHEJ). 2- Homologous recombination (HR). 2- Homologous recombination (HR).

Figure 2. DNA Lesions, Corresponding DNA Repair andFigure 2. DNA Lesions, Corresponding DNA Repair and Maintenance Systems, and Their Effect on CellularMaintenance Systems, and Their Effect on Cellular

Survival and MutagenesisSurvival and Mutagenesis

Nonhomologous end joining simply brings two ends together, Nonhomologous end joining simply brings two ends together, but bases may be lost or added as it occurs. but bases may be lost or added as it occurs.

This inaccurate process takes place mostly before replication, This inaccurate process takes place mostly before replication, in the absence of an identical copy of DNA.in the absence of an identical copy of DNA.

Associated with an elevated risk of mutagenesis, and this is Associated with an elevated risk of mutagenesis, and this is because NHEJ promotes cellular survival in the presence of because NHEJ promotes cellular survival in the presence of highly cytotoxic DSBs .highly cytotoxic DSBs .

In mammals, NHEJ is important for the repair of somatic In mammals, NHEJ is important for the repair of somatic (differentiated) cells and proliferating cells in the G1 stage.(differentiated) cells and proliferating cells in the G1 stage.

NHEJ promotes cellular survival in the presence of highly NHEJ promotes cellular survival in the presence of highly cytotoxic DSBs and may thereby enhance mutagenesis cytotoxic DSBs and may thereby enhance mutagenesis

11 - -Nonhomologous end-joining (NHEJ)Nonhomologous end-joining (NHEJ)

• NO requirement for DNA sequence similarity or complementarity

Requires specialized proteins:

Ku70/80/DNA-PKcs

Artemis

Fen1

Mre11, Rad50 and NBS

XRCC4/DNA ligase IV

Drouet, J. et al. J. Biol. Chem. 2006;281:27784-27793.

Ku (Ku70/Ku80) is thought to be the first protein to bind at each DNA end at such a double-strand break.

Ku functions as a ’toolbelt’ protein to recruit the nuclease, polymerase, and ligase activities for NHEJ, much like PCNA or the β-clamp function during DNA replication to recruit other activities

DNA single-strand breaks at *

MRN-complex may help in keeping ends together


Non Homologous End JoiningNon Homologous End Joining


DNA-PK catalytic subunit can bind well to DNA ends own its own, but its affinity is improved even further by interaction with Ku. DNA-PKcs phosphorylates the C-terminus of Artemis

The phosphorylation of the C-terminus alters the conformation of Artemis such that its C-terminal tail is no longer inhibitory for the endonucleolytic activity of Artemis. Artemis can then function to trim 5′ overhangs or 3′ overhangs endonucleolytically




Either DNA polymerase or can bind to Ku:DNA via the BRCT domains within each of these polymerases

These polymerases have specificity for gapped substrates, and have a lower fidelity than the X-family pol .

The XRCC4:DNA ligase IV complex can ligate one strand even if the other strand is not ligatable. Also the ligation can occur even when only 2 bp of annealing exists between the two ends



Takes place after replication.Takes place after replication.

Homologous recombination, acting through a series of complex DNA Homologous recombination, acting through a series of complex DNA transactions, uses the identical sister chromatid to properly align the transactions, uses the identical sister chromatid to properly align the broken ends and unerringly insert missing information.broken ends and unerringly insert missing information.

Also involved in bypassing interstrand cross-links and in repairing Also involved in bypassing interstrand cross-links and in repairing single-strand breaks (SSBs) and blocking lesions encountered during single-strand breaks (SSBs) and blocking lesions encountered during replication.replication.

HR is important for early embryogenesis and repair of proliferating HR is important for early embryogenesis and repair of proliferating cells in the S or G2 stage. cells in the S or G2 stage.

HR promotes cellular survival, but without inducing mutations. HR promotes cellular survival, but without inducing mutations.

22 - -Homologous recombination (HR)Homologous recombination (HR)

B-B- DNA adductsDNA adducts

11 - -Base excision repair (BER) is involved with small DNA adducts Base excision repair (BER) is involved with small DNA adducts (mainly oxidative and alkylating lesions), some of which may be highly (mainly oxidative and alkylating lesions), some of which may be highly

mutagenic (e.g., 7,8-dihydro-8-oxoguanine), and some cytotoxicmutagenic (e.g., 7,8-dihydro-8-oxoguanine), and some cytotoxic . .

- - BER prevents mutagenesis and promotes cellular survivalBER prevents mutagenesis and promotes cellular survival . .

- - When these lesions block elongating RNA polymerase, thenWhen these lesions block elongating RNA polymerase, then

22-- Transcription-coupled repair (TCR) removes the damage, allowing the Transcription-coupled repair (TCR) removes the damage, allowing the vital transcription to resume, which is specific to transcription- vital transcription to resume, which is specific to transcription-blocking bulky adducts, which are eliminated throughout the entire blocking bulky adducts, which are eliminated throughout the entire genome bygenome by

33--The global genome nucleotide-excision repair (GG-NER) systemThe global genome nucleotide-excision repair (GG-NER) system..

4-4-Translesional synthesis (TLS) translational Translesional synthesis (TLS) translational synthesis, a specialized, relatively error-free synthesis, a specialized, relatively error-free (but still somewhat mutagenic) and (but still somewhat mutagenic) and homologous recombination which involves homologous recombination which involves template switching and strand displacement .template switching and strand displacement .

by these two ways, the DNA damage that by these two ways, the DNA damage that blocks the regular replication machinery blocks the regular replication machinery involving DNA polymerase δ/ε (e.g., breaks involving DNA polymerase δ/ε (e.g., breaks and cross-links) can be repaired, or bypassed .and cross-links) can be repaired, or bypassed .

•Nucleotide-excision repair eliminates helix-distorting lesions in a multistep, “cut and-patch” reaction that involves more than 30 proteins.

•It has two branches:

1- Global genome repair: probes the genome for strand distortions.

2- Transcription-coupled repair: which removes distorting lesions that

block elongating RNA polymerases.

Transcription coupled repair is probably part of a broader pathway of transcription-coupled repair that includes a subgroup of nucleotide-excision–repair factors.

Nucleotide Excision Repair diseasesNucleotide Excision Repair diseases

Damage to DNA that occurs anywhere in the genome (e.g., photoproducts resulting Damage to DNA that occurs anywhere in the genome (e.g., photoproducts resulting from exposure to ultraviolet [UV] radiation) is recognized by the XPC and XPE (or from exposure to ultraviolet [UV] radiation) is recognized by the XPC and XPE (or UV-DDB) protein complexes, which are specific components of the global genome UV-DDB) protein complexes, which are specific components of the global genome nucleotide-excision repair (NER) system. nucleotide-excision repair (NER) system.

Damage that actually blocks transcription (e.g., cyclobutane pyrimidine dimers Damage that actually blocks transcription (e.g., cyclobutane pyrimidine dimers [CPDs] resulting from exposure to UV radiation) is detected by the transcription-[CPDs] resulting from exposure to UV radiation) is detected by the transcription-coupled NER system (TC-NER) system, which involves the CSB and CSA coupled NER system (TC-NER) system, which involves the CSB and CSA proteins. proteins.

The DNA helix is opened by the XPB and XPD helicases of the repair and The DNA helix is opened by the XPB and XPD helicases of the repair and transcription factor IIH (TFIIH), allowing damage verification by the XPA protein. transcription factor IIH (TFIIH), allowing damage verification by the XPA protein.

Single-strand binding protein RPA prevents reannealing, and dual incisions in the Single-strand binding protein RPA prevents reannealing, and dual incisions in the damaged strand are made by the ERCC1-XPF and XPG endonucleases, excising damaged strand are made by the ERCC1-XPF and XPG endonucleases, excising the damage as part of a piece of 25 to 30 bases. the damage as part of a piece of 25 to 30 bases.

The single-strand gap is filled by the replication machinery, and the final nick The single-strand gap is filled by the replication machinery, and the final nick sealed by DNA ligase.sealed by DNA ligase.

Figure 3. Molecular Mechanisms ofFigure 3. Molecular Mechanisms of Nucleotide-Excision RepairNucleotide-Excision Repair..

Xeroderma Pigmentosum

People with rare, inherited defects in nucleotide excision repair all have hypersensitivity to the sun that is due to defective handling of UV damage, but the clinical features are otherwise extremely heterogeneous.

The syndrome results from defects in the system of global repair, with or without deficiencies in transcription-coupled repair of distorting lesions.

Nucleotide Excision Repair diseasesNucleotide Excision Repair diseases

CockayneCockayne’’s Syndromes Syndrome

characterized by early cessation of growth and characterized by early cessation of growth and development, severe and progressive neurodysfunction development, severe and progressive neurodysfunction associated with demyelination, sensorineural hearing associated with demyelination, sensorineural hearing loss, cataracts, cachexia, and frailty.loss, cataracts, cachexia, and frailty.

Mutations in transcription-coupled repair in the genes Mutations in transcription-coupled repair in the genes encoding CSA or CSB protein cause Cockayne’s encoding CSA or CSB protein cause Cockayne’s syndrome,syndrome,

The elimination of cells with low levels of damage protects the body from cancer at the expense of promoting aging, thus revealing a trade-off between these two outcomes of DNA damage.

This phenomenon explains why there has never been a report of cancer in a person with the Cockayne syndrome, despite the repair defect and premature aging .

It indicates a clear dissociation between the outcomes of cancer and accelerated aging that result from DNA damage.

ContCont..

Has features of Cockayne’s syndrome in addition Has features of Cockayne’s syndrome in addition to brittle (unfinished) hair and nails and ichthyotic to brittle (unfinished) hair and nails and ichthyotic skin.skin.

Caused by Point mutations in the genes encoding Caused by Point mutations in the genes encoding XPB and XPD.XPB and XPD.

The helicases XPB and XPD are components of the The helicases XPB and XPD are components of the repair and transcription factor IIH (TFIIH).repair and transcription factor IIH (TFIIH).

TrichothiodystrophyTrichothiodystrophy

This model reveal a striking correlation between the degree to which This model reveal a striking correlation between the degree to which specific repair pathways are compromised and the severity of specific repair pathways are compromised and the severity of accelerated aging, strongly suggesting a causalaccelerated aging, strongly suggesting a causal

relationship.relationship.

The progressive disease in these models include many features which The progressive disease in these models include many features which highlight the importance and widespread effects of accumulating DNA highlight the importance and widespread effects of accumulating DNA damage.damage.

The life span of these mice ranges from 3 to 5 weeks to 2 years The life span of these mice ranges from 3 to 5 weeks to 2 years depending on the extent of the defect in nucleotide excision repair or depending on the extent of the defect in nucleotide excision repair or transcription coupled repair.transcription coupled repair.

Informative mouse models of nucleotideInformative mouse models of nucleotideexcision repair syndromesexcision repair syndromes

Both the mice with TrichothiodystrophBoth the mice with Trichothiodystrophyy and the and the mice with Cockayne’s syndrome mice with Cockayne’s syndrome have low rates have low rates of spontaneous cancerof spontaneous cancer, a finding that is , a finding that is consistent with the idea that the defect in consistent with the idea that the defect in transcription coupled repair provides protection transcription coupled repair provides protection against carcinogenesis.against carcinogenesis.

ContCont..

Most mutations and large genomic alterations that are relevant to Most mutations and large genomic alterations that are relevant to cancer originate from DNA injury or aberrant genome maintenance:cancer originate from DNA injury or aberrant genome maintenance:

1- For instance, repair of double-strand breaks involves extensive 1- For instance, repair of double-strand breaks involves extensive phosphorylation of histone H2AX followed by modification of phosphorylation of histone H2AX followed by modification of histones by ubiquitination and sumoylation, all of which are histones by ubiquitination and sumoylation, all of which are required for efficient double-strand break repair.required for efficient double-strand break repair.

2- Repair of single-strand breaks involves the formation of large chains 2- Repair of single-strand breaks involves the formation of large chains of poly(adenosine diphosphate [ADP]–ribose) on target proteins by of poly(adenosine diphosphate [ADP]–ribose) on target proteins by the enzyme poly (ADP–ribose) polymerase (PARP); the chains of the enzyme poly (ADP–ribose) polymerase (PARP); the chains of poly (ADP–ribose) probably serve as a platform to recruit the poly (ADP–ribose) probably serve as a platform to recruit the appropriate mechanism of repair.appropriate mechanism of repair.

DNA Damage and DNA MaintenanceDNA Damage and DNA Maintenance in Cancerin Cancer

Cells may also induce DNA injury for protection against cancer

How this can be happen?

•With every round of DNA replication, a number of protective telomeric repeats are lost at the ends of chromosomes because

the telomerase enzyme that adds new repeats is silenced in most somatic cells.

•Clonal outgrowth of a precancerous cell results in critically short telomeres that behave similarly to double-strand breaks, awakening the DNA damage response system and triggering

cell-cycle arrest and cell death.

Jan H.J. Hoeijmakers, Ph.D. MOLECULAR ORIGINS OF CANCER DNA Damage, Aging, and Cancer Student: Alia...

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