DNA REPAIRANAND C.R.

MSc. BIOTECHNOLOGYCUSAT

DNA DAMAGE

Wood Smoke Causes Significant DNA Damage

Polycyclic Aromatic Hydrocarbons, (PAH) adhere to DNA

Wood smoke particles • generate free radicals

• cause lipid peroxidation,

• DNA damage• NFkappaB activation

• TNF-alpha release in macrophages.

Types of DNA DamageAll four of the bases in DNA (A, T, C, G) can be covalently modified at various positions.

Mismatches of the normal bases because of a failure of proofreading during DNA replication.

Breaks in the backbone.

Crosslinks or Covalent linkages can be formed between bases

Direct DNA Damage

Some agents damage DNA directly

Chemicals and light• Chemicals - alkylating agents • Methyl and ethyl groups added to DNA bases• This type of damage can be repaired by direct reversal

involving special enzymes• They remove the offending atoms and restore the base

• Ultraviolet rays

Random photons of ultraviolet (UV) light induce aberrant bonding between neighbouring pyrimidines (thymine & cytosine) bases on the same strand of DNA. The will prevent the replication machine from duplicating the DNA. The cell will die!

This type of defect can be readily reversed by a process called photoreactivation. Visible light energy is used to reverse the defect (in bacteria, yeasts, protists, some plants, and some animals but NOT in humans)

Other forms of DNA damage

DeaminationAn amino group of Cytosine is removed

and the base becomes Uracil An amino group of Adenine is removed

and the base becomes HypoxanthineAn amino group of Guanine is removed

and the base becomes Hypoxanthine

Other forms of DNA damage

• Depurination - the base is simply ripped out of the DNA molecule leaving a gap (like a missing tooth)…

Repairing Damaged Bases

1. Direct Reversal of Base Damage2. Excision Repair

1. Base Excision Repair (BER)2. Nucleotide Excision Repair (NER)3. Mismatch Repair (MMR)

DNA Repair Pathways

Direct Reversal • The simplest of the human DNA repair

pathways • most energy efficient method• involves the direct reversal of the highly

mutagenic alkylation lesion O6-methylguanine (O6-mG)

• Carried out by the product of the MGMT gene O6-alkylguanine DNA alkyltransferase (AGT) (O6-methylguanine DNA methyltransferase)

• Correction of the lesion occurs by direct transfer of the alkyl group on guanine to a cysteine residue in the active site of MGMT in a "suicide" reaction.

• The inactivated alkyl-MGMT protein is then degraded in an ATP-dependent ubiquitin proteolytic pathway.

• The O6-mG adduct is generated in low levels by the reaction of cellular catabolites with the guanine residues in the DNA.

• A number of DNA-damaging chemotherapeutic agents attack the O6 position on guanine, forming the most potent cytotoxic DNA adducts known

• AGT activity correlates inversely with sensitivity to agents that form such O6-alkylguanine DNA adducts

.

Gerson S L JCO 2002;20:2388-2399

Mechanism of action of AGT inhibition by O6-benzylguanine (BG). BG penetrates the active site pocket of AGT where it comes in contact with the sulfur of cysteine 145. A covalent transfer reaction inactivates the protein

DNA-damaging chemotherapeutic

agents• Carmustine (BCNU)• Temozolomide• Streptozotocin• Dacarbazine.

carmustine

Temozolomide

Streptozotocindacarbazine

O6 alkylation by temozolomide and carmustine (BCNU).

The methylating agent temozolomide forms O6-methylguanine DNA adducts that induce cell death by invoking mismatch repair.

The chloroethylating agent BCNU initially forms O6-chloroethylguanine DNA adducts that then rearrange to a 1,6-ethanoguanine cyclic intermediate followed by a crosslink with the cytosine directly on the opposite strand.

Base excision repair (BER)

Multi-step process that corrects non-bulky damage to bases– Oxidation– Methylation– Deamination– spontaneous loss of the DNA base– significant threat to genome fidelity and stability

• BER has two subpathways: – short patch: replaces the lesion with a single

nucleotide– long patch: replaces the lesion with

approximately 2 to 10 nucleotides• Both initiated by the action of a DNA

glycosylase that cleaves the N-glycosidic bond between the damaged base and the sugar phosphate backbone of the DNA.

DNA repair by base excision

• A base-specific DNA glycosylase detects an altered base and removes it

• AP endonuclease and phosphodiesterase remove sugar phosphate.

• DNA Polymerase fills and DNA ligase seals the nick

Nucleotide Excision

Same as Base Excision Except that– It recognizes more varieties of damage– Remove larger segments of DNA (10 -100s of

bases)

Nucleotide excision repair a large multienzyme

compound scans the DNA strand for anomalities

upon detection a nuclease cuts the strand on both sides of the damage

DNA helicase removes the oligonucleotide

the gap is repaired by DNA polymerase and DNA ligase enzymes

Mismatch Repair (MMR)Mismatch repair deals with correcting

mismatches of the normal bases; that is, failures to maintain normal base pairing (A ･ T,

C ･ G)Recognition of a mismatch requires several

different proteins including one encoded by MSH2.

Cutting the mismatch out also requires several proteins, including one encoded by MLH1.

How does the MMR system know which is the incorrect nucleotide?

In E. coli, certain adenines become methylated shortly after the new strand of DNA has been synthesized.

The MMR system if detects a mismatch, it assumes that the nucleotide on the already-methylated (parental) strand is the correct one and removes the nucleotide on the freshly-synthesized daughter strand.

How such recognition occurs in mammals is not yet known.

Mismatch Repairing Mechanism

Proteins involved in the DNA repairing of E. coli.

Recombinational Repair

• This type of repair is much more complicated than is excision repair, and requires many more gene products. The products of a number of these repair genes are induced by radiation damage, and therefore this type of repair requires protein synthesis before it can function. Because of its complexity, this type of repair makes mistakes

Postreplication Repair (recombinational DNA repair)

(a) The dots indicate lesions in the DNA.

(b) DNA synthesis proceeds up to a lesion and then skips past the lesion, leaving a gap in the daughter strand.

(c) Filling of the daughter strand gaps with DNA from parental strands by a recombinational process that requires a functional recA gene.

(d) Gaps in the parental strands are repaired by repair replication

Recombinational Excision Repair

• This process occurs in the part of the chromosome that was replicated prior to irradiation, i.e., where two sister duplexes were present before irradiation.

• After the excision of the lesion, the resulting gap is fi lled by the same recombinational process

The recombinational repair of excision gaps in E. coli.

a) UV radiation-induced lesions are produced in both the replicated and unreplicated portions of the genome

b) The gaps produced by excision in the unreplicated portion are repaired by the classical methods of nucleotide excision repair 

c) The gaps produced in the replicated portion of the chromosome are repaired by a recombinational process that requires both recA and recF (C-D)

Genetic Diseases• Defects in the repair system lead to permanent DNA damage causing xeroderma pigmentosum and other genetic diseases.

Xeroderma Pigmentosum• Defects in one of seven genes

(XPA-XPG) important in repairing DNA damage caused by ultraviolet (UV) light.

• Recessive genetic disorder• At a young age

– Multiple basal cell carcinomas (basaliomas)

– other skin malignancies• Most common causes of death

– metastatic malignant melanoma– squamous cell carcinoma

Pathophysiology• Defect in (NER)• Seven xeroderma pigmentosum repair

genes, XPA through XPG, have been identified.

• In addition to the defects in the repair genes, UV-B radiation also has immunosuppressive effects that may be involved in the pathogenesis of xeroderma pigmentosum

Midwest: 20 min sunlight would kill 50% of cells in culture dish

2%-10% UVB reaches basal layer of skin

Leffell and Brash, Scientific American, July 1996

• Xeroderma pigmentosum variant– The defect in this condition is not in NER,

but is instead in postreplication repair– A mutation occurs in DNA polymerase έ

• Several immunologic abnormalities have been described in the skin of patients with xeroderma pigmentosum

• Clinical studies of the skin of patients indicate prominent depletion of Langerhans cells induced by UV radiation

Other defects in cell-mediated immunity – impaired cutaneous responses to recall

antigens– decreased ratio of circulating T-helper cells

to suppressor cells– impaired lymphocyte proliferative

responses to mitogen– impaired production of interferon in

lymphocytes– reduced natural killer cell activity.

XP patients

• At high risk for skin cancer• Must be protected from the sun and

other sources of UV radiation• With great caution in sun exposure, XP patientscan live to middle age

RaceCases of xeroderma pigmentosum are

reported in persons of all races.

SexAn equal prevalence has been reported in males

and females.

Age The disease is usually detected at age 1-2 years.

Cockayne Syndrome

• Rare autosomal recessive, heterogeneous, multisystem disorder

• Named after English physician Edward Alfred Cockayne

• Characterized by:– Dwarfism– progressive pigmentary retinopathy– birdlike facies– photosensitivity

Forms of Cockayne syndrome

• CS Type I, the classic form– characterized by normal fetal growth with the onset of

abnormalities in the first two years of life. – Impairment of vision, hearing, and the central and

peripheral nervous system progressively degenerate until death in the first or second decade of life.

• CS Type II, otherwise known as connatal CS– involves very little neurological development after birth. – Death usually occurs by age 7. – Has also been designated as COFS syndrome

• subdivided into several conditions (COFS type 1, 2, 3 (which is itself is associated with Xeroderma Pigmentosum) and type 4).

• CS Type III –rare and characterized by late onset–milder than Type I and II.

• Xeroderma-pigmentosum-Cockayne syndrome (XP-CS) –occurs when an individual also suffers

from Xeroderma pigmentosum–Some symptoms of each disease are expressed.

Genetics

• Mutations in the ERCC6 and ERCC8 genes

• The proteins made by these genes are involved in repairing damaged DNA via the transcription-coupled repair mechanism, particularly the DNA in active genes.

• If either the ERCC6 or the ERCC8 gene is altered, DNA damage is not repaired. As this damage accumulates, it can lead to malfunctioning cells or cell death.

Fanconi Anemia• Fanconi anemia is one of the inherited

anemias that causes bone marrow failure.• It is a recessive disorder.• There are at least 11 different mutations

causing fanconi anemia.– A*, B, C, D1, D2, E, F, G, I, J, and L.

• It is considered mainly a blood disease.• Many patients eventually develop acute

myelogenous leukemia at an early age.• Patients are very likely to develop

squamous cell carcinomas.

Clinical Manifestations• Fanconi Anemia characterized by

• physical abnormalities

• bone marrow failure

• increased risk of malignancy.

• Physical abnormalities

1. short stature

2. abnormalities of the thumbs, forearms, skeletal system, eyes, kidneys and urinary tract, ear, heart, gastrointestinal system, oral cavity, and central nervous system

Physical abnormalities contd.

3. Hearing loss

4. Hypogonadism

5. Developmental delay

Progressive bone marrow failure with pancytopenia typically presents in the first decade, often initially with thrombocytopenia or leukopenia

Diagnosis and Treatment• Patients are usually smaller than average.• Blood tests may show a low WBC, RBC, and

platelet count.• Fatigue.• Frequent infections.• Frequent nosebleeds• Easy bruising. • Treatments include:

– Bone marrow transplant.– Growth factors.

• Hematopoietic (blood-stimulating) growth factors are used to stimulate WBC production.

– Androgens.• Male hormones often stimulate the production

of RBCs and platelets.

Ataxia-telangiectasia• Rare childhood disease that aff ects

the brain and other parts of the body.

• Ataxia refers to uncoordinated movements, such as walking.

• Telangiectasias are enlarged blood vessels (capil laries) just below the surface of the skin.

• Telangiectasias appear as ti ny red spider-l ike veins.

Causes, incidence, and risk factors

• Ataxia-telangiectasia is inherited, which means it is passed down through families- Autosomal recessive trait.

• The disease results from defects in the ataxia telangiectasia mutated (ATM) gene. • Defects in this gene can lead to abnormal cell death in various places of the body,

including the part of the brain that helps coordinate movement.• Male and female are equally affected.

ComplicationsCancer such as lymphoma

Diabetes

Kyphosis: forward curving of the spine

Progressive movement disorder that leads to wheelchair use

Scoliosis : spine is curved from side to side

Severe, recurrent lung infections

Health Comments

•increased cancer risk

•accelerated aging

•neurodegenerative diseases

Inefficient or incorrect DNA repair due to

micronutrient shortages leads to genome

instability

Since degenerative diseases are partly caused by DNA damage, it makes sense to diagnose and nutritionally

prevent the underlying cause, genome instability.

– A knowledge of optimal intakes for vitamins and minerals that are needed to prevent DNA damage is required

– "Excessive genome instability, a fundamental cause of disease, is often an indication of micronutrient deficiency and is therefore preventable

– accurate diagnosis of genome instability using DNA damage biomarkers that are sensitive to micronutrient deficiency is technically feasible

– it should be possible to optimise nutritional status and verify efficacy by diagnosis of a reduction in genome damage rate after intervention"

zSUMMARY

REFERENCES• http://www.rndsystems.com/

mini_review_detail_objectname_MR03_DNADamageResponse.aspx

• http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0002369/

• http://www.suite101.com/content/defects-in-dna-repair-and-resulting-diseases-a137961

• Essential cell biology, 2/e ALBERTS BRAY• GENES IX BENJAMIN LEWIN

THANK YOU