DNA Repair

DNA can be damaged by external agents and by replication errors. Since maintenance of the correct base sequence of DNA and of daughter DNA molecules is essential for hereditary fidelity, repair systems have evolved that restore the correct base sequence.

Mismatch Repair and Methylation of DNA:

• DNA polymerases occasionally catalyze incorporation of an "incorrect" base that cannot form a hydrogen bond with the template base in the parental strand; such errors usually are corrected by the editing function of these enzymes. 
• The editing process occasionally fails, so a second system, called mismatch repair, exists for correcting the errors that are not edited out. In mismatch repair, a pair of non-hydrogen-bonded bases (e.g., G . . . T) within a helix are recognized as aberrant and a polynucleotide segment of the daughter strand is excised, thereby removing one member of the unmatched pair. 
• The resulting gap is filled in by pol I, which presumably uses this "second chance" to form correct base pairs; then the final seal is made by DNA ligase. If it is only to correct and not create errors, the mismatch repair system must be capable of distinguishing the correct base in the parental strand from the incorrect base in the daughter strand. 
• Rare methylated bases (methyl-A and methyl-C) provide the basis for this distinction. In E. coli, an enzyme, DNA methylase, methylates adenine in the sequence GATC. Methylation occurs soon (but not immediately) after the replication fork has synthesized such a sequence in the daughter strand. Thus, the nucleotides in the daughter strand are usually not methylated near the fork, whereas those in the parental strand are always completely methylated. 
• The mismatch repair system recognizes the degree of methylation of a strand and preferentially excises nucleotides from the undermethylated strand. The daughter strand is always the undermethylated strand, so that parental information is retained.

Glycosylases:
Occasionally, uracil or other incorrect bases might become incorporated into new DNA strands. These bases are generally removed by a pathway that begins with the cleavage of the N-glycosidic bond by an enzyme called a glycosylase. Many glycosylase enzymes are known, and each is base-specific (e.g., uracil N-glycosylase). This enzyme cleaves the N-glycosidic bond and leaves the deoxyribose in the backbone. A second enzyme (AP endonuclease) makes a single cut, freeing one end of the deoxyribose. AP stands for apurinic acid, a polynucleotide from which purines have been removed by hydrolysis of the N-glycosidic bonds. This step is followed by removal of the deoxyribose and several adjacent nucleotides (probably by a second enzyme that acts at the other side of the apurinic site), after which pol I fills the gap with correct nucleotides. This sequence, endonuclease-enlargement polymerase, is an example of a general repair mechanism called excision repair.

Repair of Cytosine deamination

Alterations of DNA Molecules:

Several agents can break phosphodiester bonds. Among the more common are peroxides and various metal ions (e.g., Fez+, Cu2+). Ionizing radiation also efficiently produces strand breaks. DNases present in cells probably also sometimes break phosphodiester bonds. Double-strand breakage, i.e., two single-strand breaks opposite one another, occurs because of exposure to all forms of ionizing radiation. Single-strand breaks can be repaired by DNA ligases, although sometimes additional enzymes are needed. Double-strand breaks rarely are repaired. Bases can be changed into different compounds by a variety of chemical and physical agents. For instance, ionizing radiation can break purine and pyrimidine rings and can cause several types of chemical substitutions, the most common being in guanine and thymine. The best studied altered base is the dimer occurred by covalent linkage of two adjacent pyrimidine rings; these are produced by ultraviolet (UV) irradiation. The most prevalent of these dimmers is the thymine dimer. The main effects of the presence of thymine dimers are the following:

1. The DNA helix becomes distorted as the thymines, which are in the same strand, are pulled toward one another.
2. As a result of the distortion, hydrogen bonding to adenines in the opposite strand is significantly weakened, causing inhibition of advance of the replication fork.

Thymine dimer

General Mechanisms for Repair of DNA:

Repair of damaged bases was first observed and is best understood in bacteria. It is a widespread and probably universal phenomenon in both prokaryotes and eukaryotes. Some systems that repair dimers repair other types of DNA damage also. Four major pathways for DNA repair exist that can be
subdivided into two different classes: light-induced repair (photoreactivation) and light-independent repair (dark repair). The latter can be accomplished by three distinct mechanisms:
1. Excision of the damaged nucleotides (excision repair).
2. Reconstruction of a functional DNA molecule from undamaged fragments (recombinational repair).
3. Disregard of the damage (SOS repair).

Photoreactivation:
Photoreactivation is a light-induced (300-600 nm) enzymatic cleavage of a thymine dimer to give two thymine monomers. It is done by photolyase, an enzyme that acts on dimers present in single- and doublestranded DNA. The enzyme-DNA complex absorbs light and uses the photon energy to cleave specific C-C bonds of the cyclobutylthymidine dimer. Photolyase is also active against cytosinethymine dimers and cytosine dimers, which are also occurred by UV irradiation but much less frequently.

Excision Repair:

• Excision repair is a multistep enzymatic process. Several mechanisms are known, but only two will be explained. All require an early incision step, in which a nuclease recognizes the distortion produced by a thymine dimer and makes a cut in the sugar-phosphate backbone. 
• Following this, a DNA polymerase mediates a strand displacement step. In E. coli, two cleavages can be occurred; the first is 12 nucleotides from the 5' side of the dimer and the second is 4-5 nucleotides from the 3' side. Each cut produces a 3'-OH and a 5'-Phosphate group. 
• The T-OH group of the first cut is recognized by pol I, which then synthesizes a new strand, displacing the dimer-containing DNA strand. When the second cut is reached, the displaced fragment falls away and DNA ligase enzyme joins the 3'-OH and 5'-Phosphate groups. 
• In Micrococcus luteus, the first step is cleavage of the N-glycosidic bond of the thymine at the 5' end of the dimer (by a dimer-specific glycosylase), leaving a free deoxyribose which is removed, leaving a free 3'-OH group. 
• As in E. coli, pol I polymerizes from this end and displaces the dimer-containing strand. After the dimer has been displaced, a second cut is made, the displaced strand falls away, and ligase forms the final phosphodiester bond. Repair of dimers in mammalian cells is more complicated and poorly understood, and requires a larger collection of enzymes.

Base excision repair mechanism

Recombination Repair:

• Recombination repair is a mechanism for forming a functional DNA molecule from two damaged molecules. It is an important repair process for dividing cells because a replication fork may arrive at a damaged site, such as a thymine dimer, before the excision repair system has eliminated damage. 
• When pol III reaches a thymine dimer, an adenine is added to the growing strand. However, the distortion of the helix caused by the dimer weakens the hydrogen bond and activates the polymerase editing function, and the adenine is removed. 
• The cycle begins again-an adenine is added and then removed-the net result of which is that the replication fork fails to advance. A cell in which DNA synthesis is permanently stalled cannot complete a round of replication and does not divide. However, in a way that is not understood, after a pause of ~5 seconds per dimer, chain growth begins again beyond the thymine dimer block. 
• The result of this process is that the daughter strands have large gaps, one for each unexcised thymine dimer. Viable daughter cells cannot be produced by continued replication alone because the strands having the thymine dimer will continue to turn out defective daughter strands and the first set of daughter strands would be fragmented when the growing fork enters a gap. However, by a recombination mechanism called sister-strand exchange proper double stranded molecules can be made. 
• The essence of sister-strand exchange is that a single stranded segment free of any defects is excised from an undamaged strand on the homologous DNA segment at the replication fork and somehow inserted into the gap created by the thymine dimer. 
• The combined action of polymerase I and DNA ligase joins this inserted piece to adjacent regions, thus filling in the gap. The gap formed in the donor molecule is also repaired. If this exchange and gap filling are done for each thymine dimer, two complete single daughter strands can be formed, and each can serve in the next round of replication as a template for synthesis of normal DNA molecules. 
• The system fails if two dimers in opposite strands are very near one another because no undamaged segments are available. Since recombinational repair occurs after DNA replication, in contrast with excision repair, it is often called postreplicational repair.

SOS Repair:

SOS repair includes a bypass system that allows DNA chain growth across damaged segments at the cost of fidelity of replication. It is an error-prone process; even though intact DNA strands are formed, the strands are often altered. As described above, activation of the editing system stalls replication at a thymine dimer. In SOS repair, the editing system is relaxed to allow polymerization to proceed across a dimer. Relaxation of the editing system means a loss of the ability to remove "incorrect" bases added to the growing strand. Most of the time, pol III inserts two adenines at a dimer site. However, the distortion increases the error frequency, allowing other nucleotides to be added to the chain. This error-prone repair is the major cause of UV-induced mutagenesis. An important nuclear protein, conserved from yeast to mammals, is the Ku heterodimer. This protein binds to DNA and repairs double strand breaks caused by x-rays and other agents. The Ku heterodimer is essential in maintaining chromosome integrity.
Human Diseases and DNA Repair Deficiency:

• Human disease may result from inability to carry out certain stages of DNA repair. The best studied disease, xeroderma pigmentosum, is because of mutations in genes that encode the UV excision system. 
• Cells cultured from tissue obtained from affected individuals are killed by much smaller doses of UV light than are normal cells. Furthermore, the removal of thymine dimers in DNA from these cells is very inefficient. People with this disease develop skin lesions when exposed to sunlight and commonly develop one of several kinds of skin cancer. 
• Ataxia telangiectasia is characterized by severe abnormalities in various organ systems and a high incidence of lymphoreticular cancer. Defective DNA repair was suspected when patients developed an unexpected severe or fatal reaction while undergoing radiotherapy for cancer. As predicted, nontumor cells cultured from these patients are hypersensitive to x-rays. 
• Fanconi's syndrome, a lethal aplastic anemia, is also due to defective DNA repair. Cells from affected persons cannot repair interstrand cross-links or damage induced by x-rays. Two premature aging disorders (HutchinsonGilford syndrome and Bloom's syndrome) and several other disorders (Cockayne's syndrome and retinoblastoma) are also associated with defects in DNA repair. 
• Cells from patients with some chromosome abnormalities (e.g., Down syndrome) may also show aberrant DNA repair. Several human DNA mismatch repair genes are associated with hereditary nonpolyposis colon cancer (HNPCC). 
• One of these mismatch repair genes (hMSH2) is located on the short arm of chromosome 2; others are located on chromosome 3. Defects in any of these DNA repair genes make individuals susceptible to colon cancer as well as to other cancers.