Chapter Four

DNA Repair
4.1 INTRODUCTION

All cellular macromolecules are susceptible to undergo damage. If RNA's or Proteins undergo damage they can rapidly be degraded and resynthesized via transcription and translation.

But, damage to DNA, genetic material leads to mutation and subsequently changes lethargic to organism.

4.2 DNA DAMAGE

DNA damage can be described in terms of following points

Spontaneous and inherited gene mutations Natural cell processes External causes

4.2.1 Spontaneous and inherited gene mutations

The building block of DNA are nucleotides. These nucleotides are arranged in a specific order inside a gene.

During the course of cell reproduction if the sequence of these nucleotides get altered i.e., a nucleotide is inserted or deleted the whole reading frame for aminoacids changes leading to formation of different protein. This process is reffered as Spontaneous mutation.

If these changes occur in germ cell (egg or sperm) these mutation can be inherited by the next generation.

4.2.2 Natural cell processes

Production of energy in a living cell occurs by the process known as cellular oxidation. In addition to energy, a lot of toxic biproducts are also produced during the process. These are a class of free radicals which can damage DNA as well as cellular proteins and

fats. 4.2.3 External causes

Nucleotide sequences in DNA can be altered by physical or chemical agents. Physical agents includes Ultraviolet light, X-rays and other ionizing radiations. Chemical agents causing mutation include toxins like benzo[a]pyrene, medications like those used in chemotherapy, and most deadly poison, cigarette smoke.

4.3 EFFECT OF DNA DAMAGE ON CELL CYCLE

DNA damage checkpoints sense DNA damage both before the entry of cell into S phase (a G1 checkpoint) as well as after S phase (a G2 checkpoint).

Damage to DNA before the cell enters S phase inhibits the action of Cdk2 thus stopping the progression of the cell cycle until the damage can be repaired.

If the damage is so severe that it cannot be repaired, the cell self-destructs by apoptosis. Damage to DNA after S phase (the G2 checkpoint), inhibits the action of Cdk1 thus preventing the cell from proceeding from G2 to mitosis.

Defects in checkpoint control have been seen in certain hereditary cancer syndromes and at early stages of cell transformation.

Mutations in checkpoint control genes therefore may contribute to the genetic instability that appears to drive neoplastic evolution.

After damage DNA needs to be repair to ensure the integrity of the cellular genome. This is accomplished using multienzyme, multipathway system.

4.4 DNA REPAIR MECHANISM

There are four major DNA repairing mechanisms :

Base Excision Nucleotide Excision Mismatch Repair Repairing Strand Breaks

4.4.1 Base excision

DNA base may be modified by deamination or dealkylation. The position of the damaged base is called abasic site or AP site. In base excision repair, an altered base is removed from AP site by a DNA glycosylase enzyme, followed by excision of the resulting sugar phosphate.

The small gap left in the DNA helix is filled in by the sequential action of DNA polymerase and DNA ligase.

4.4.2 Nucleotide Excision

While the base excision repair machinery can recognize specific lesions in the DNA and can correct only damaged bases that can be removed by a specific glycosylase, the nucleotide excision repair enzymes recognize bulky distortions in the shape of the DNA double helix.

In E.Coli, proteins UvrA, UvrB, and UvrC are involved in removing the damaged nucleotides. In yeast, the proteins similar to Uvr's are named RADxx ("RAD" stands for "radiation"), such as RAD3, RAD10. Etc.

Removal of a short single stranded DNA segment includes the lesion, creates a single-strand gap in the DNA, which is subsequently filled in by DNA polymerase, which uses the undamaged strand as a template.

4.4.3 Mismatch repair

Most mismatches are due to replication errors. However, mismatches can also be produced by other mechanisms for example, by deamination of 5-methyl cytosine to produce thymidine improperly paired to G.

Regardless of the mechanism by which they are produced, mismatches can always be repaired by the mismatch repair pathway.

To repair mismatched bases, the system has to know which base is the correct one. In E. coli this is accomplished by methylation of all adenines that occur within 5' GATC sequence.

Methylation is caused by the activity of an enzyme called DAM methylase (DNA adenine methylase).

Immediately after replication the template strand is methylated but the newly synthesized strand is not methylated yet. This allows E. coli cells to distinguish between old strand (correct) and newly synthesized strand (incorrect).

The repairing process begins with the protein MutS which binds to mismatched base pairs. The MutS-MutL complex activates MutH. The activation of MutH cleaves unmethylated strand at GATC site.

Subsequently, the segment from the cleavage site to the mismatch is removed by exonuclease.

The gap is then filled by DNA polymerase III and DNA ligase.

4.4.4 Repairing Strand Breaks

Ionizing radiations and certain chemicals can produce both single strand breaks (SSB's) and double strand breaks (DSB's) in the DNA backbone.

Single Strand Breaks (SSB's) : Breaks in single strand of DNA molecule are repaired using

same enzyme system as used in Base-Excision Repair (BER).

Double Strand Breaks (DSB's) : There are two mechanisms by which cell attempts to

repair a complete break in a DNA molecule.

Direct joining of broken ends. This is accomplished using a protein that recognises and bind to exposed ends and bring them together for ligation. They would prefer to see some complementary nucleotides but can proceed without them so this type of joining is also called Nonhomologous End-Joining(NHEJ). A protein called Ku is essential for non-homologous joining. Ku is a heterodimer consisting of two subunits namely Ku70 and Ku80.

Homologous Recombination: Here the broken ends are repaired using the information on the intact sister chromatid (available in G2 after chromosome

duplication), or on the homologous chromosome (in G1, i.e., before each chromosome has been duplicated). This requires searching around in the nucleus for the homolog or on the same chromosome if there are duplicate copies of the gene on chromosome oriented in same direction or opposite directions. Two of the proteins used in homologous recombination are encoded by the genes BRCA1 and BRAC2.

4.5 DNA REPAIR AND AGING

Some scientists believe that the accumulation of uncorrected DNA damage over years is a major cause of aging. They cite the following observations:

Animals with the fastest rates of DNA repair generally have the longest life spans. Animals with the highest rates of oxidative damage by free radicals (and specifically, with oxidative damage to DNA) generally have the shortest life spans.

Exposure to external causes of DNA damage (ultraviolet light, tobacco) decreases life span.

Humans who have genetic diseases resulting in greater spontaneous DNA damage or inefficient DNA repair often show signs of premature aging.

4.6

DNA REPAIR DISORDERS

The Ataxia telegiectaxia, Xeroderma pigmentosum(XP), Fanconi's anemia, Bloom syndrome are associated with defects in DNA repair.

Ataxia telegiectaxia is caused by lack or inactivation of ATM protein kinase which mobilises the cellular response to double strand breaks in the DNA.

Xeroderma pigmentosum is a rare inherited disease of humans which among other things, predispose the patients to pigmented lesions and an elevated risk of skin cancer. XP can be caused by mutation of gene that plays a role in nucleotide excision repair leading to a

reduction in or elimination of Nucleotide Excision Repair.

Fanconi anemia (FA) is an autosomal recessive disorder caused by defects in at least eight distinct genes FANCA, B, C, D1, D2, E, F and G. The clinical phenotype of all FA complementation groups is similar and is characterized by progressive bone marrow failure, cancer proneness and typical birth defects.

The gene that causes Bloom's Syndrome is DNA helicase, RecQ-like, type 2 gene(BLM gene).It has not yet been established how mutations in this gene cause the symptoms seen in Bloom's Syndrome, but it is believed that the absence of the BLM gene product probably destabilizes other enzymes that participate in DNA replication and repair.

4.7 MEDICINE AND DNA REPAIR MODULATION

There exists a vast body of evidence that correlates DNA damage with death and disease. Increasing the activity of some DNA repair enzyme can add many healthy and disease-free years to an aging population

4.7.1 Cancer treatment

The hall mark of cancer is continious cell division. Cell division requires, DNA replication, transcription and translation of many genes needed for growth.

That means controling the activity of enzymes involved in DNA repair can inhibit the spread of cancer.

Procedures such as chemotherapy and radiotherapy work by overwhelming the capacity of the cell to repair DNA damage ultimately resulting in cell death.

The side effect is that other noncancerous but similarly rapidly dividing cells such as stem cells in the bone marrow are also affected.

Modern cancer treatments attempt to localize the DNA damage to cells and tissues only associated

with cancer. 4.7.2 Gene therapy

For therapeutic uses of DNA repair, the challenge is to discover which particular DNA repair enzymes exhibit the most precise specificity for damaged sites, so its overexpression will lead to enhanced DNA repair function.

Once the appropriate repair factor has been identified the next step is finding the appropriate way to incorporate them into viable cell to generate viable disease and ageing treatment.

Gene repair refers to the form of gene therapy which targets and repair chromosomal mutation responsible for a disorder.

It does so by replacing the altered DNA sequence with the desired sequence, using techniques such as oligonucleotide directed mutagenesis.

4.8 DNA REPAIR AND EVOLUTION

DNA repair is critical for the maintenance of genome integrity and replication fidelity in all cells.

Rarely DNA damage is not repaired or repaired at wrong place leading to change in sequence of nucleotides. If such a mutation occur in germ cell it will lead to production of gamete possesing mutated gene sequence which will ultimately pass on to offspring.

The rate of evolution in a particlar species is a function of rate of mutation. Therefore, rate and accuracy of DNA repair plays an important role in evolution of new species.