Question

Describe the multiple mechanisms of DNA damage surveillance and repair. If these exist, how do mutations still arise?

Answer 1

DNA damage, if unrepaired, may result in mutation or cell death. Therefore, cells have evolved complex signaling networks to carefully monitor the integrity of the genome during DNA replication, and to initiate cell cycle arrest, repair, or apoptotic responses if errors are detected.

DNA damage surveillance:

There are numerous strategies with inherent advantages and disadvantages that may be used for the evaluation of DNA damage and repair. DNA is the primary target following exposure to stimuli such as ultraviolet (UV) radiation, DNA alkylators, certain environmental carcinogens, oxidative stress and chemotherapeutic drugs. All these damaging factors produce lesions on DNA and a base alteration promoting a break in the DNA helix . Double-strand breaks (DSBs) are lethal to cells, as they affect both strands of DNA and promote the loss of genetic information . DNA damage, which frequently occurs in eukaryotic cells, may promote genomic instability and aid the development of disease, including cancer . Following DNA damage, cellular responses are induced and allow the cell to repair the damage or process the damage via a variety of mechanisms . Therefore, DNA repair proteins are important biomarkers for predicting the response of tumors to genotoxic stress and the prognosis of patients with more accuracy.

PCR is one of the most frequently used techniques for detecting DNA damage . DNA amplification is stopped at the sites of damage via the blocking of the progression of Taq polymerase, which results in a decrease in the quantity of PCR product and a reduced number of DNA templates, which do not contain the Taq-blocked lesions as they are not amplified . This is considered to be a simple and reliable method in which particular segments of DNA are specifically replicated and visualized using agarose gels that resolve a range of DNA fragments (50–50,000 bp) dependent on the agarose percentage.

The comet assay, also known as single-cell gel electrophoresis, is simple and is considered to be one of the gold standard methods for measuring DNA strand breaks (single or double) in eukaryotic cells. In addition to being a method for detecting DNA breaks, it is also possible to detect UV-induced pyrimidine dimers, oxidized bases and alkylation damage following the introduction of lesion-specific endonucleases.

ELISA is one of the most commonly used immunological methods for the quantification of DNA damage and consists of affixing an unknown quantity of antigen to a surface and applying an unknown quantity of antibody to the surface so that the antibody binds to the antigen. The antibody is linked to an enzyme that may be quantified via the addition of an appropriate substrate (colored, fluorescent or radioactive).

Oxidative stress and absorption of UV light by nucleic acids has been established to be one of the causes of oxidative DNA damage, which may promote cancer development . The improvement of HPLC coupled to tandem MS with an electrospray ionization mode, may be a sensitive and accurate method to detect modified bases of the oxidative-damaged DNA and UV-induced dimeric pyrimidine photoproducts .

DNA damage repair:

While the cell is able to evolve into either an apoptotic or senescent state, these actions are performed as a last resort. For each type of DNA damage, the cell has evolved a specific method of repairing the damage or eliminating the damaging compound.

O6-Methylguanine DNA methyltransferase (MGMT; DNA alkyltransferase) cleaves both methyl and ethyl adducts from guanine bases on the DNA structure. The reaction is not a catalytic (enzymatic) reaction but is stoichiometric (chemical), consuming one molecule of MGMT for each adduct removed. Cells that have been engineered to overexpress MGMT are more resistant to cancer, likely because they are able to negate a larger amount of alkylating damage. A recent study by Niture, et al., reports an increase in MGMT expression by use of cysteine/glutathione enhancing drugs and natural antioxidants.

DNA polymerases such as polymerase-δ contain proofreading activities and are primarily involved in replication error repair. When an error is detected, these polymerases halt the process of DNA replication, work backward to remove nucleotides from the daughter DNA chain until it is apparent that the improper nucleotide is gone, and then reinitiate the forward replication process. Mice with a point mutation in both copies of the Pold1 gene demonstrated a loss of proofreading activity by DNA polymerase-δ and developed epithelial cancers at a significantly higher rate than did mice with wild-type genes or with a single copy mutation.

A group of proteins known as mismatch excision repair (MMR) enzymes is capable of correcting errors of replication not detected by the proofreading activities of DNA polymerase. MMR enzymes excise an incorrect nucleotide from the daughter DNA and repair the strand using W-C pairing and the parent DNA strand as the correct template.This is especially crucial for errors generated during the replication of microsatellite regions, as the proofreading activity of DNA polymerase does not detect these errors. To a lesser degree, MMR enzymes also correct a variety of base pair anomalies resulting from DNA oxidation or alkylation. These mutations include modified base pairs containing O6-methylguanine and 8-oxoguanine, and carcinogen and cisplatin adducts.Mutations in the human mismatch excision repair genes MSH2 and MLH1 are associated with hereditary non-polyposis colorectal cancer (HNPCC) syndrome.

Base excision repair (BER) involves multiple enzymes to excise and replace a single damaged nucleotide base. The base modifications primarily repaired by BER enzymes are those damaged by endogenous oxidation and hydrolysis. A DNA glycosylase cleaves the bond between the nucleotide base and ribose, leaving the ribose phosphate chain of the DNA intact but resulting in an apurinic or apyrimidinic (AP) site. 8-Oxoguanine DNA glycosylase I (Ogg1) removes 7,8-dihydro-8-oxoguanine (8-oxoG), one of the base mutations generated by reactive oxygen species. Polymorphism in the human OGG1 gene is associated with the risk of various cancers such as lung and prostate cancer. Uracil DNA glycosylase, another BER enzyme, excises the uracil that is the product of cytosine deamination, thereby preventing the subsequent C→T point mutation. N-Methylpurine DNA glycosylase (MPG) is able to remove a variety of modified purine base.

Nucleotide excision repair (NER) repairs damage to a nucleotide strand containing at least 2 bases and creating a structural distortion of the DNA. NER acts to repair single strand breaks in addition to serial damage from exogenous sources such as bulky DNA adducts and UV radiation.  The same pathway may be used to repair damage from oxidative stress.

Double-strand breaks in DNA can result in loss and rearrangement of genomic sequences. These breaks are repaired by either nonhomologous end-joining (NHEJ) or by homologous recombination (HR), also called recombinational repair or template–assisted repair.

Mutation:

All cells possess DNA-repair enzymes that attempt to minimize the number of mutations that occur. These enzymes work in two ways. Some are pre-replicative and search the DNA for nucleotides with unusual structures, these being replaced before replication occurs; others are post-replicative and check newly synthesized DNA for errors, correcting any errors that they find . A possible definition of mutation is therefore a deficiency in DNA repair.

DNA repair processes exist in both prokaryotic and eukaryotic organisms, and many of the proteins involved have been highly conserved throughout evolution. In fact, cells have evolved a number of mechanisms to detect and repair the various types of damage that can occur to DNA, no matter whether this damage is caused by the environment or by errors in replication. Because DNA is a molecule that plays an active and critical role in cell division, control of DNA repair is closely tied to regulation of the cell cycle. (Recall that cells transit through a cycle involving the G₁, S, G₂, and M phases, with DNA replication occurring in the S phase and mitosis in the M phase.) During the cell cycle, checkpoint mechanisms ensure that a cell's DNA is intact before permitting DNA replication and cell division to occur. Failures in these checkpoints can lead to an accumulation of damage, which in turn leads to mutations.

Defects in DNA repair underlie a number of human genetic diseases that affect a wide variety of body systems but share a constellation of common traits, most notably a predisposition to cancer . These disorders include ataxia-telangiectasia (AT), a degenerative motor condition caused by failure to repair oxidative damage in the cerebellum, and xeroderma pigmentosum (XP), a condition characterized by sensitivity to sunlight and linked to a defect in an important ultraviolet (UV) damage repair pathway. In addition, a number of genes that have been implicated in cancer, such as the RAD group, have also been determined to encode proteins critical for DNA damage repair.