DNA damage,cellular sensing/responding,

and repair

Rebecca Fry, Ph.D.

DNA damage

DNA damage, due to environmental factors and normal metabolic processes inside the cell, occurs at a rate of 1,000 to 1,000,000 molecular lesions per cell per day.

DNA Damage

While this constitutes only 0.000165% of the human genome's approximately 6 billion bases (3 billion base pairs)…

unrepaired lesions in critical genes (such as tumor suppressor genes) can impede a cell's ability to carry out its function and appreciably increase the likelihood of tumor formation.

Failure to repair DNA lesions may result in blockages of transcription and replication, mutagenesis, and/or cellular cytotoxicity.

In humans, DNA damage has been shown to be involved in a variety of genetically inherited disorders, in aging, and in carcinogenesis.

Sources of DNA Damage

DNA damage can be subdivided into two main types:

ENDOGENOUS and EXOGENOUS

Endogenous sources of DNA Damage

endogenous damage such as attack by reactive oxygen species produced from normal metabolic byproducts

Types of Damage

The main types of damage to DNA due to endogenous cellular processes:

1. oxidation of bases [e.g. 8-oxo-7,8-dihydroguanine (8-oxoG)] In living cells ROS are formed continuously as a consequence of metabolic and other biochemical reactions . These ROS include superoxide (O2

–·), hydrogen peroxide (H2O2), hydroxyl radicals (OH·) and singlet oxygen (1O2)

2. alkylation of bases (usually methylation), such as formation of 7-methylguanine, 1-methyladenine, O6 methylguanine

3. hydrolysis of bases, such as deamination, depurination and depyrimidination.

4. "bulky adduct formation" (i.e. benzo[a]pyrene diol epoxide-dG adduct).

5. mismatch of bases, due to errors in DNA replication, in which the wrong DNA base is stitched into place in a newly forming DNA strand, or a DNA base is skipped over or mistakenly inserted.

Exogenous sources of DNA damage

caused by external agents such as

• ultraviolet [UV 200-300nm] radiation from the sun

• other radiation frequencies, including x-rays and gamma rays

• human-made mutagenic chemicals• cancer chemotherapy and radiotherapy

What is the difference between DNA damage and

mutation??

It is important to distinguish between DNA damage and mutation, the two major types of error in DNA.

DNA damage and mutation are fundamentally different.

Damage is a physical abnormality in the DNA, such as single and double strand breaks, 8-hydroxydeoxyguanosine residues and polycyclic aromatic hydrocarbon adducts.

In contrast to DNA damage, a mutation is a change in the base sequence of the DNA. A mutation cannot be recognized by enzymes once the base change is present in both DNA strands, and thus a mutation cannot be repaired.

At the cellular level, mutations can cause alterations in protein function and regulation.

Mutations are replicated when the cell replicates. In a population of cells, mutant cells will increase or decrease in frequency according to the effects of the mutation on the ability of the cell to survive and reproduce.

What are the types of DNA damage??

1. Base loss2. Base modification3. Replication errors

4. Inter-strand X-links5. DNA-protein X-links

6. Strand Breaks

The bond linking DNA bases with deoxyribose is labile under physiological conditions.

Within a typical mammalian cell, several thousand purines and several hundred pyrimidines are spontaneously lost per diploid genome per day.

Loss of a purine or pyrimidine base creates an apurinic/apyrimidinic (AP) site (also called an abasic site):

1. Base loss

2. Base modification2a. DeaminationThe primary amino groups of nucleic acid

bases are somewhat unstable. The amino group is removed from the amino acid and converted to ammonia.

In a typical mammalian cell, about 100 uracils are generated per haploid genome per day in this fashion.

Other deamination reactions include conversion of adenine to hypoxanthine, guanine to xanthine, and 5-methyl cytosine to thymine.

Example: cytosine deamination                                       

Spontaneous deamination is the hydrolysis reaction of cytosine into uracil, releasing ammonia in the process.

2. Base modification2b. Chemical modificationThe nucleic acid bases are susceptible to

numerous modifications by a wide variety of chemical agents.

For example, several types of hyper-reactive oxygen (singlet oxygen, peroxide radicals, hydrogen peroxide and hydroxyl radicals) are generated as byproducts during normal oxidative metabolism and also by ionizing radiation (X-rays, gamma rays). These are frequently called Reactive Oxygen Species (ROS). ROS can modify DNA bases. A common product of thymine oxidation is thymine glycol:

Another type of chemical modification: methylation/alkylation

• Many environmental chemicals, including "natural" ones (frequently in the food we eat) can also modify DNA bases, frequently by addition of a methyl or other alkyl group (alkylation).

• In addition, normal metabolism frequently leads to alkylation.

• It has been shown that S-adenosylmethionine, the normal biological methyl group donor, reacts accidentally with DNA to produce alkylated bases like 3-methyladenine at a rate of several thousand per day per mammalian diploid genome.

2. Base modification2b. Photodamage

Ultraviolet light is absorbed by the nucleic acid bases, and the resulting influx of energy can induce chemical changes.

• The most frequent photoproducts are the consequences of bond formation between adjacent pyrimidines within one strand, and, of these, the most frequent are cyclobutane pyrimidine dimers (CPDs).

Ultraviolet light induces the formation of covalent linkages by reactions localized on the C=C double bonds

3. Replication errorsAnother major source of potential

alterations in DNA is the generation of mismatches or small insertions or deletions during DNA replication.

Although DNA polymerases are moderately accurate, and most of their mistakes are immediately corrected by polymerase-associated proofreading exonucleases, nevertheless the replication machinery is not perfect.

4. Inter-strand crosslinksBy attaching to bases on both

strands, bifunctional alkylating agents such as the psoralens can cross-link both strands.

Cross-links can also be generated by UV and ionizing radiation.

5. DNA-protein crosslinksDNA topoisomerases generate covalent

links between themselves and their DNA substrates during the course of their enzymatic action.

Usually these crosslinks are transient and are reversed as the topoisomerase action is completed. Occasionally something interferes with reversal, and a stable topoisomerase-DNA bond is established.

Bifunctional alkylating agents and radiation can also create crosslinks between DNA and protein molecules. All of these lesions must be repaired.

6. Strand breaksSingle-strand and double-strand

breaks are produced at low frequency during normal DNA metabolism by topoisomerases, nucleases, replication fork "collapse", and repair processes.

Breaks are also produced by ionizing radiation.

What can the cell do to protect itself?

DNA damage is recognized by sensor proteins that then initiate a network of signal transduction pathways.

This ultimately results in the activation of effector proteins that execute the functions of the DNA damage response, including recruitment of DNA repair proteins, cell cycle arrest, damage induced transcription, or the induction of apopotosis.

DNA damage recognition

An option: DNA damage checkpoints

• After DNA damage, cell cycle checkpoints are activated.

• Checkpoint activation pauses the cell cycle and gives the cell time to repair the damage before continuing to divide.

• The cell cycle of eukaryotic cells can be divided into four successive phases:

• M phase (mitosis), in which the nucleus and the cytoplasm divide;

• S phase (DNA synthesis), in which the DNA in the nucleus is replicated,

• two gap phases, G1 and G2.

• The G1 phase is a critical stage, allowing responses to extracellular cues that induce either commitment to a further round of cell division or withdrawal from the cell cycle (G0) to embark on a differentiation pathway.

Cell Cycle

The transition from one phase of the cell cycle to the next is controlled by cyclin–CDK (cyclin-dependent kinase) complexes which ensure that all phases of the cell cycle are executed in the correct order.

• DNA damage checkpoints occur at the G1/S and G2/M boundaries. An intra-S checkpoint also exists.

• Checkpoint activation is controlled by two master kinases, ATM and ATR.

• ATM responds to DNA double-strand breaks and disruptions in chromatin structure, whereas ATR primarily responds to stalled replication forks.

• These kinases phosphorylate downstream targets in a signal transduction cascade, eventually leading to cell cycle arrest.

DNA damage checkpoints

• A class of checkpoint mediator proteins including BRCA1, MDC1, and 53BP1 has also been identified.

• These proteins seem to be required for transmitting the checkpoint activation signal to downstream proteins.

• p53 is an important downstream target of ATM and ATR, as it is required for inducing apoptosis following DNA damage.

What happens if we have defective ATM??

ataxia telangiectasia mutated

Ataxia-telangiectasia is a rare, childhood neurological disorder that causes degeneration in the part of the brain that controls motor movements and speech.

Its most unusual symptom is an acute sensitivity to ionizing radiation, such as X-rays or gamma-rays. The first signs of the disease, which include delayed development of motor skills, poor balance, and slurred speech, usually occur during the first decade of life.

Disease: Ataxia-telangiectasia

Telangiectasias (tiny, red "spider" veins), which appear in the corners of the eyes or on the surface of the ears and cheeks, are characteristic of the disease, but are not always present and generally do not appear in the first years of life.

About 20% of those with A-T develop cancer, most frequently acute lymphocytic leukemia or lymphoma.

Many individuals with A-T have a weakened immune system, making them susceptible to recurrent respiratory infections.

ATM mutations are associated with breast

cancer• Researchers have found that having a mutation in one copy of the ATM gene in each cell (particularly in people who have at least one family member with ataxia-telangiectasia) is associated with an increased risk of developing breast cancer.

• About 1 percent of the United States population carries one mutated copy of the ATM gene in each cell. These genetic changes prevent many of the body's cells from correctly repairing damaged DNA.

So thank goodness for DNA Repair

DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome.

In human cells, both normal metabolic activities and environmental factors such as UV light and Radiation can cause DNA damage, resulting in as many as 1 million individual molecular lesions per cell per day.

Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes.

DNA repair mechanisms

Cells cannot function if DNA damage corrupts the integrity and accessibility of essential information in the genome (but cells remain superficially functional when so-called "non-essential" genes are missing or damaged).

Depending on the type of damage inflicted on the DNA's double helical structure, a variety of repair strategies have evolved to restore lost information.

Direct reversal

Cells are known to eliminate damage to their DNA by chemically reversing it.

These mechanisms do not require a template, since the types of damage they counteract can only occur in one of the four bases.

Such direct reversal mechanisms are specific to the type of damage incurred and do not involve breakage of the phosphodiester backbone.

An example:

methylation of guanine bases, is directly reversed by the protein methyl guanine methyl transferase (MGMT), the bacterial equivalent of which is called as ogt.

This is an expensive process because each MGMT molecule can only be used once; that is, the reaction is stoichiometric rather than catalytic.

How do we end up with methylated Guanine???

Exposure to alkylating agents!

O6-meG can mispair with thymine G/C to A/T transitions

Damage induced mimics some chemotherapeutics

Damage induced mimics environmental exposures

guanine cytosine

thymine

Can be cytotoxic or mutagenic lesion

X

Using genomics to PREDICT population responses to exposures

Can we use gene expression levelsto predict responses to DNA alkylating agents?

Fry et al, Genes and Development 2008

NIH PDR Cell Lines Represent Healthy Genetically Diverse Population

450 healthy, unrelated individuals

24 lymphoblastoid cell lines

European (120)European (120)

Native (30)Native (30)

African (120)African (120)Mexican (60)Mexican (60)

Asian (120)Asian (120)

Training Population

ResistantResistantSensitiveSensitive

Establish Range of Sensitivity in Cells Exposed to MNNG (0.5 ug/ml)

Training Population

Establish training population based on extreme responders

ResistantResistantSensitiveSensitive

Alkylation-Sensitivity-Associated Gene Sets Identified

Statistically Significant Differential Expression 1.5 FC , p-value < 0.05

ResistantResistantSensitiveSensitive

% Control growthExpre

ssio

n inte

nsi

ty

Statistically significant association (p<0.01) of %

control growth and expression

ResistantResistantSensitiveSensitive

0

50

100

150

200

250

0 20 40 60 80 100

ResistanceResistance

SensitivitySensitivity

6 4 9 20 12 8 22 7

-1 0 +1

48 genes 39 genes

Basal Treated Ratio

121 genes

94% accuracy

Apply two-class prediction algorithm:SVM to 16 cell lines of test population

High expression in MNNG resistant cells

High

6 4 9 20 12 8 22 7

-1 0 +1

ResistantResistantSensitiveSensitive

Low

48 genes

2 genes

The top hit: high expression in MNNG resistant cells

ResistantResistant

SensitiveSensitive

The most significant positive association of MGMT expression with resistance

MGMT

inactive inactive

1. Base excision repair (BER), which repairs damage to a single base caused by oxidation, alkylation, hydrolysis, or deamination.

The damaged base is removed by a DNA glycosylase, resynthesized by a DNA polymerase, and a DNA ligase performs the final nick-sealing step.

These hydrolyze the N-glycosylic bond between the base and the deoxyribose, as illustrated here by the action of uracil DNA N-glycosylase:

2. Nucleotide excision repair (NER), which recognizes bulky, helix-distorting lesions such as pyrimidine dimers and 6,4 photoproducts.

A specialized form of NER known as transcription-coupled repair deploys NER enzymes to genes that are being actively transcribed.

NER involves the following steps:

• Damage recognition• Binding of a multi-protein complex at the

damaged site• Double incision of the damaged strand several

nucleotides away from the damaged site, on both the 5' and 3' sides

• Removal of the damage-containing oligonucleotide from between the two nicks

• Filling in of the resulting gap by a DNA polymerase

• Ligation

What happens if we have defective NER??

Xeroderma pigmentosum (XP)

Xeroderma pigmentosa, or XP, is an autosommal ressessive genetic disorder of DNA repair in which the ability to repair damage caused by ultraviolet (UV) light is deficient (NER deficiency).

This disorder leads to multiple basal cell carcinomas (basaliomas) and other skin malignancies at a young age.

In severe cases, it is necessary to avoid sunlight completely.

The two most common causes of death for XP victims are metastatic malignant melanoma and squamous cell carcinoma.

Cockayne syndrome is a rare autosomal recessive congenital disorder characterized by growth failure, impaired development of the nervous system, abnormal sensitivity to sunlight (photosensitivity), and premature aging.

3. Mismatch repair (MMR), which corrects errors of DNA replication and recombination that result in mispaired (but undamaged) nucleotides.

Are there health effects from MMR deficiency?

Double-strand breaks

Double-strand breaks (DSBs), in which both strands in the double helix are severed, are particularly hazardous to the cell because they can lead to genome rearrangements.

Various mechanisms exist to repair DSBs:

non-homologous end joining (NHEJ), recombinational repair (also known as template-assisted repair or homologous recombination repair

In NHEJ

DNA Ligase IV, a specialized DNA Ligase that forms a complex with the cofactor XRCC4, directly joins the two ends.

DNA ligase, shown above repairing chromosomal damage, is an enzyme that joins broken nucleotides together by catalyzing the formation of an internucleotide ester bond between the phosphate backbone and the deoxyribose nucleotides.

Recombinational repair requires the presence of an identical or nearly identical sequence to be used as a template for repair of the break.

The enzymatic machinery responsible for this repair process is nearly identical to the machinery responsible for chromosomal crossover during meiosis.

This pathway allows a damaged chromosome to be repaired using a sister chromatid (available in G2 after DNA replication) or a homologous chromosome as a template.

Recombinational Repair

Translesion synthesisTranslesion synthesis is a DNA damage tolerance process that allows the DNA replication machinery to replicate past DNA lesions such as thymine dimers or AP sites.

It involves switching out regular DNA polymerases for specialized translesion polymerases (e.g. DNA polymerase V), often with larger active sites that can facilitate the insertion of bases opposite damaged nucleotides.

The polymerase switching is thought to be mediated by, among other factors, the post-translational modification of the replication processivity factor PCNA.

Translesion synthesis polymerases often have low fidelity (high propensity to insert wrong bases) relative to regular polymerases.

PCNA

Proliferating Cell Nuclear Antigen

DNA repair and cancer

Inherited mutations that affect DNA repair genes are strongly associated with high cancer risks in humans.

Hereditary nonpolyposis colorectal cancer (HNPCC) is strongly associated with specific mutations in the DNA mismatch repair pathway.

BRCA1 and BRCA2, two famous mutations conferring a hugely increased risk of breast cancer on carriers, are both associated with a large number of DNA repair pathways, especially NHEJ and homologous recombination.

Modern cancer treatments attempt to localize the DNA damage to cells and tissues only associated with cancer, either by physical means (concentrating the therapeutic agent in the region of the tumor) or by biochemical means (exploiting a feature unique to cancer cells in the body).

Cancer ChemotherapyThe hallmark of all cancers is continuous cell

division.

Each division requires both the replication of the cell's DNA (in S phase) and

transcription and translation of many genes needed for continued growth.

So, any chemical that damages DNA has the potential to inhibit the spread of a cancer.

Many (but not all) drugs used for cancer therapy do their work by damaging DNA.

Cyclophosphamide Cytoxan®

alkylating agents; form interstrand and/or intrastrand crosslinks

Melphalan Alkeran®

Busulfan Myleran®

Chlorambucil Leukeran®

Mitomycin Mutamycin®

Cisplatin Platinol® forms crosslinks

Bleomycin Blenoxane® cuts DNA strands between GT or GC

Irinotecan Camptosar®inhibit the proper functioning of enzymes (topoisomerases) needed to

unwind DNA for replication and transcriptionMitoxantrone Novantrone®

Dactinomycin Cosmegen® inserts into the double helix preventing its unwinding

The table lists (by trade name as well as generic name) some of the anticancer drugs that specifically target DNA.

Some questions

• How many DNA repair proteins are there in humans?

• What about conservation across species??