Biochemistry, DNA Repair

Fundamentals

DNA damage plays an integral part in the development of cancer and other diseases. The mediators of DNA damage are both exogenous and endogenous. Endogenous damage occurs within the cell as a result of normal cellular processes such as errors caused during DNA replication such as incorrect nucleotide insertion or mismatches, DNA instability resulting from depurination (cleavage of bonds between the deoxyribose sugar and the purine base) or due to free radicals formed during oxidation-reduction reactions such as deamination of DNA and proteins. 

Most of these errors are corrected by DNA polymerase during replication with its inherent proofreading mechanism via its 3'-exonuclease activity. This mechanism allows for the polymerase to read the most recently placed base before adding the next one. If the base does not pair correctly with the base on the template strand, then the exonuclease mechanism of DNA pol III cuts the phosphodiester bond holding that section of the strand and releases the nucleotide. This process is not perfect, however, and incorrect bases are commonly left in the newly synthesized DNA strand. Mismatch repair proteins correct this by locating incorrectly matched nucleotides and excising them before placing the correct nucleotide in its place.

Exogenous damage, also called induced damage, is the result of external factors acting on the cell. The common causes of exogenous damage include Radiation such as UV light exposure (UVA and UVB) and ionizing radiations, thermal disruptions, toxins, and drugs that cause base-pair mismatches, intercalation causing inhibition of replication. UV damage causes the cross-linking of adjacent cytosine and thymine bases, forming harmful dimers.

Ionizing radiations such as X-rays can dissociate water molecules in the cell, producing hydroxyl radicals that can react with DNA, altering its structure and cause DNA strands to break. Certain toxins and drugs cause chemical associations resulting in single or double-strand breakage in the DNA helix. Thermal disruption via DNA being exposed to high temperatures causes higher rates of depurination to occur, as well as single-strand breaks. Radiation exposure also causes strand breaks. These types of induced damage are very severe and can cause irreparable damage and harm to the cell. Similarly, environmental toxins such as cigarette smoke contain carcinogens like benzo-pyrene. These chemicals can be oxidized by cellular enzymes and cause DNA damage by forming DNA adducts.

Damage inflicted on the DNA during or after replication and transcription are not to be confused with mutations. DNA damage results in single and double-strand breakages and is repairable by the different actions of enzymes in the cell. This is because the enzymes have a template for repair present in either the complementary strand of a damaged DNA molecule or a homologous chromosome that can be copied and used to implement the correct base pairs and base orders in the double helix.

Mutation, on the other hand, is a permanent alteration to the base pair sequence of the host's DNA. It is irreparable to enzymes as mutation leaves no template for the correct base pair sequence to be integrated into the strand. Mutation can also cause changes in how certain genes are expressed, in contrast to damage, which causes either a halt in gene function or is deleterious to the cell. Damage may lead to mutation, but while mutation may lead to harmful byproducts, it does not cause direct damage to the mutated gene or DNA molecule.[2][3][4]

Mechanism

There are many types of repair that are specific to certain types of damage inflicted on a DNA molecule and focus on ssDNA or dsDNA.

Single strand DNA (ssDNA) repair mechanisms include mismatch repair, base excision repair, and nucleotide excision repair.[5]

1. Mismatch repair is a type of correcting mechanism that fixes mistakes in DNA after replication or transcription that remain uncorrected by the exonuclease function of DNA pol III, specifically bases that incorrectly match with the wrong base on the complementary strand. Mismatch repair proteins correct this by locating incorrectly matched nucleotides and excising them via its own exonuclease system before placing the correct nucleotide in its place with DNA polymerase and sealing the nick created from the excision with DNA ligase. This process is a common repair mechanism for endogenous damage, as replication errors fall under this category. Mutations in genes for proteins responsible for mismatch repairs such as MLH1, MSH2, MSH6, and PMS2 can result in certain cancers such as hereditary non-polyposis colorectal cancer (also known as Lynch Syndrome) and ovarian cancers.[6]
2. Base excision repair works by excising a single damaged base or nucleotide; this is under the mediation of DNA glycosylases cutting the bond between the nitrogenous base and the deoxyribose sugars. When the glycosylases remove the base, it creates an apurinic site if the removed base was a purine (adenine, guanine), or an apyrimidinic site if the excised base was a pyrimidine (cytosine, thymine). The phosphodiester backbone of the DNA strand is then nicked by endonucleases and DNA polymerase removes the damaged nucleotide and replaces it with the correct one. Spontaneous deamination of cytosine frequently occurs in human DNA and, if left unrepaired, causes mutations.[7]
3. Nucleotide excision repair is most common when exposure to UV radiation has caused the formation of thymine or cytosine dimers. It usually involves localized DNA problems such as mismatched bases or adducts (as happens with benzopyrene exposure). These dimers can be extremely harmful to a DNA molecule and usually lead to cell death or cancerous developments. These are the leading cause of melanomas. Nucleotide excision repair differs from the repairs mentioned above as it excises a large swath of nucleotides upstream and downstream of the dimerized bases and resynthesizes the whole region with the polymerase. Specific repair endonucleases excise the problem area, and deoxyribonucleotides fill the gap with the help of DNA polymerase. A DNA ligase ligates the newly synthesized fragment to the original DNA strand. Xeroderma pigmentosum is a condition where the nucleotide excision repair mechanism is defective due to mutation in the genes that encode these repair proteins.[8]

Double strand DNA (dsDNA)  damage is much harder to repair because the damaged helix has no template from which to copy and replace the afflicted bases and nucleotides. Two major pathways can alleviate issues involving double-stranded breaks: homologous recombination and non-homologous end-joining.

1. Homologous recombination is a useful mechanism for a double-stranded break as it takes advantage of the homologous sister chromatid or chromosome that has a DNA sequence that is identical to the damaged strand. This identical sequence serves as the template for repair and enzymes similar to those used in crossover during meiosis act to repair the broken strands.[9]
2. Non-homologous end-joining is essential before the broken DNA replicates, as a double-strand break usually leads to cell death. It uses micro homologies, which are short homologous sequences on the end of individual strands of DNA, to join the two fractured ends back together by translocation. However, if there are base deletions at the break site, the rejoining of the strands can lead to mutations in the genome. Harmful X-rays or carcinogens can cause such breaks, resulting in the gross rearrangement of chromosomes. Burkitt’s lymphoma is an example of cancer arising due to translocation. BRCA1 and BRCA2 proteins are involved in the homologous repair, and mutations in these genes are known to cause breast cancer.[10]

Clinical Significance

DNA repair is crucial to correcting the mistakes our replicative machinery makes when forming new materials within our bodies. Damage to DNA molecules, if left unrepaired, can often lead to carcinogenesis, which is the development of cancer cells in the affected area that can easily spread and replicate through mitosis. Conversely, cancer developments can be understood and analyzed through the mechanisms in which DNA damage occurs, and repair processes are affected as a result of the carcinogens in the body. This process has received extensive study relative to exogenous damage such as ionizing radiation that can lead to leukemia and melanoma due to various exposures, ranging from simple ultraviolet to X-ray. 

Novel methods are under development to target DNA repair pathways that have suffered mutagenetic effects and use them to fight the tumor to induce malignant cell death. DNA repair can be recruited to the carcinogens benefit, so the goal of this area of research is to turn the repair pathways back on the tumor and induce its degradation.[11][12][13][14]