Top 7 DNA Damage Repair Mechanisms (With Diagram)

This article throws light upon the top seven DNA damage repair mechanisms.

The top seven DNA damage repair mechanisms are: (1) Proofreading by DNA Polymerase (2) Excision Repairs (3) Spontaneous Repair by DNA Double Helix (4) Homologous and Non-homologous DNA Repair (5) SOS Repair (6) Mismatch Repair of Single-Base Mispairs and (7) Photo-reactivation UV-induced Pyrimidine Dimers.

Mechanism # 1. Proofreading by DNA Polymerase:

Because the specificity of nucleotide addition by DNA polymerases is determined by Watson- Crick base pairing, a wrong base (e.g., A instead of G) occasionally is inserted during DNA synthesis. Indeed, a subunit of E. coli DNA polymerase III introduces about 1 incorrect base in 104 inter-nucleotide linkages during replication in vitro. Since an average E. coli gene is about 103 bases long, an error frequency of 1 in 10⁴ base pairs would cause a potentially harmful mutation in every tenth gene during each replication, or 10^-1 mutations per gene per generation.

However, the measured mutation rate in bacterial cells is much less, about 1 mistake in 10⁹ nucleotide polymerization events or, equivalently, 10^-5 to 10^-6 mutations per gene per generation (assuming ~1000 base pairs per gene). This increased accuracy in vivo is largely due to the proofreading function of E. coli DNA polymerases. An experiment demonstrated that the 3′ → 5′ exonuclease activity of E. coli DNA polymerase I can remove a mismatched base at the 3′ growing end of a synthetic primer-template complex. In DNA polymerase III, this function resides in the e subunit of the core polymerase.

When an incorrect base is incorporated during DNA synthesis, the polymerase pauses, and then transfers the 3′ end of the growing chain to the exonuclease site where the mispaired base is removed. Then the 3′ end is transferred back to the polymerase site, where this region is copied correctly. Proofreading is a property of almost all bacterial DNA polymerases. Both the 8 and e DNA polymerases of animal cells, but not the polymerase, also have proofreading activity. It seems likely that this function is indispensable for all cells to avoid excessive genetic damage.

Mechanism # 2. Excision Repairs:

There are multiple pathways for DNA repair, using different enzymes that act upon different kinds of lesions. Two of the most common pathways are shown in figure 10.3 A, B. In both, the damage is excised, the original DNA sequence is restored by a DNA polymerase that uses the undamaged strand as its template, and the remaining break in the double helix is sealed by DNA ligase (Fig. 10.2).

As shown, DNA ligase uses a molecule of ATP to activate the 5′ end at the nick (step 1) before forming the new bond (step 2). In this way, the energetically unfavorable nick- sealing reaction is driven by being coupled to the energetically favorable process of ATP hydrolysis.

The two pathways differ in the way in which the damage is removed from DNA. The first pathway, called base excision repair, involves a battery of enzymes called DNA glycosylases, each of which can recognize a specific type of altered base in DNA and catalyze its hydrolytic removal. There are at least six types of these enzymes, including those that remove deaminated Cs, deaminated As, different types of alkylated or oxidized bases, bases with opened rings, and bases in which a carbon-carbon double bond has been accidentally converted to a carbon-carbon single bond.

As an example of the general mechanism of base excision repair, the removal of a deaminated C by uracil DNA glycosylase is shown in figure. 10.3 A. It is thought that an altered base is detected when DNA glycosylases travel along DNA using base-flipping to evaluate the status of each base pair. Once a damaged base is recognized, the DNA glycosylase reaction creates a deoxyribose sugar that lacks its base. This “missing tooth” is recognized by an enzyme called AP endonuclease, which cuts the phosphodiester backbone, and the damage is then removed and repaired.

Depurination, which is by far the most frequent type of damage suffered by DNA, also leaves a deoxyribose sugar with a missing base. Depurinations are directly repaired beginning with AP endonuclease, following the bottom half of the pathway.

The second major repair pathway is called nucleotide excision repair. This mechanism can repair the damage caused by almost any large change in the structure of the DNA double helix. Such “bulky lesions” include those created by the covalent reaction of DNA bases with large hydrocarbons (such as the carcinogen benzopyrene), as well as the various pyrimidine dimers (T-T, T-C, and C-C) caused by sunlight. The DNA helix distortions are recognized by the UvrABC endonuclease, a multi-subunit enzyme encoded by the three genes uvrA, uvrB, and uvrC.

This enzyme makes one cut in the damaged DNA strand, the phosphodiester backbone of the abnormal strand is cleaved on both sides of the distortion, and an oligonucleotide containing the lesion is peeled away from the DNA double helix by a DNA helicase enzyme. The large gap produced in the DNA helix is then repaired by DNA polymerase I and sealed by DNA ligase (Fig. 10.3 B).

Mechanism # 3. Spontaneous Repair by DNA Double Helix:

The double-helical structure of DNA is ideally suited for repair because it carries two separate copies of all the genetic information-one in each of its two strands. Thus, when one strand is damaged, the complementary strand retains an intact copy of the same information, and this copy is generally used to restore the correct nucleotide sequences to the damaged strand.

The nature of the bases also facilitates the distinction between undamaged and damaged bases. Thus, every possible deamination event in DNA yields an unnatural base, which can therefore be directly recognized and removed by a specific DNA glycosylase.

An indication of the importance of a double-stranded helix to the safe storage of genetic information is that all cells use it; only a few small viruses use single-stranded DNA or RNA as their genetic material. The chance of a permanent nucleotide change occurring in these single-stranded genomes of viruses is thus very high.

Mechanism # 4. Homologous and Non-homologous DNA Repair:

A potentially dangerous type of DNA damage occurs when both strands of the double helix are broken, leaving no intact template strand for repair. Breaks of this type are caused by ionizing radiation, oxidizing agents, replication errors, and certain metabolic products in the cell. If these lesions were left unrepaired, they would quickly lead to the breakdown of chromosomes into smaller fragments. However, two distinct mechanisms have evolved to restructure the potential damage.

The simplest to understand is non-homologous end-joining, in which the broken ends are juxtaposed and rejoined by DNA ligation, generally with the loss of one or more nucleotides at the site of joining (Fig. 10.4) This end-joining mechanism, which can be viewed as an emergency solution to the repair of double-strand breaks, is a common outcome in mammalian cells.

Although a change in the DNA sequence (a mutation) results at the site of breakage, so little of the mammalian genome codes for proteins that this mechanism is apparently an acceptable solution to the problem of keeping chromosomes intact.

The specialized structure of telomeres prevents the ends of chromosomes from being mistaken for broken DNA, thereby preserving natural DNA ends. An even more effective type of double-strand break repair exploits the fact that cells that are diploid contain two copies of each double helix.

In this second repair pathway, called homologous end-joining, general recombination mechanisms come into play that transfer nucleotide sequence information from the intact DNA double helix to the site of the double-strand break in the broken helix. This type of reaction requires special recombination proteins that recognize areas of DNA sequence matching between the two chromosomes and bring them together.

A DNA replication process then uses the undamaged chromosome as the template for transferring genetic information to the broken chromosome, repairing it with no change in the DNA sequence. In cells that have replicated their DNA but not yet divided, this type of DNA repair can readily take place between the two sister DNA molecules in each chromosome; in this case, there is no need for the broken ends to find the matching DNA sequence in the homologous chromosome. Such type of repair is called as recombination repair.

Mechanism # 5. SOS Repair:

Cells have evolved many mechanisms that help them survive in an unpredictably hazardous world. Often an extreme change in a cell’s environment activates the expression of a set of genes whose protein products protect the cell from the deleterious effects of this change. One such mechanism shared by all cells is the heat-shock response, which is evoked by the exposure of cells to unusually high temperatures. The induced “heat-shock proteins” include some that help stabilize and repair partly denatured cell proteins.

Cells also have mechanisms that elevate the levels of DNA repair enzymes, as an emergency response to severe DNA damage. The best-studied example is the so-called SOS response in E. coli. The SOS response is a post replication DNA repair system that allows DNA replication to bypass lesions or errors in the DNA. The SOS uses the RecA protein. The signal (thought to be an excess of single-stranded DNA) first activates the RecA protein. The RecA protein, stimulated by single-stranded DNA, is involved in the activation of the LexA thereby inducing the response. It is an error-prone repair system.

During normal growth, the SOS genes are negatively regulated by LexA repressor protein dimers. Under normal conditions, LexA binds to a 20 bp consensus sequence (the SOS box) in the operator region for those genes. Activation of the SOS genes occurs after DNA damage by the accumulation of single stranded (ssDNA) regions generated at replication forks, where DNA polymerase is blocked. RecA forms a filament around these ssDNA regions in an ATP-dependent fashion, and becomes activated. The activated form of RecA interacts with the LexA to facilitate the LexA self-cleavage from the operator (Fig. 10.5).

Once the LexA proteins are cleaved then no repression of the SOS genes occurs. Operators that bind LexA weakly are the first to be fully expressed. In this way LexA can sequentially activate different mechanisms of repair. Genes having a weak SOS box (such as lex A, recA, uvrA, uvrB, and uvrD) are fully induced in response to even weak SOS-inducing treatments. Thus the first SOS repair mechanism to be induced is nucleotide excision repair (NER), whose aim is to fix DNA damage without commitment to a full-fledged SOS response.

Cells have an additional mechanism that helps them respond to DNA damage: they delay progression of the cell cycle until DNA repair is complete. For example, one of the genes expressed in response to the E. coli SOS signal is sulA, which encodes an inhibitor of cell division. Thus, when the SOS functions are turned on in response to DNA damage, a block to cell division extends the time for repair. When DNA repair is complete, the expression of the SOS genes is repressed, the cell cycle resumes, and the undamaged DNA is segregated to the daughter cells.

While this “error-prone” DNA repair can be harmful to individual bacterial cells, it is presumed to be advantageous in the long term because it produces a burst of genetic variability in the bacterial population that increases the likelihood of a mutant cell arising that is better able to survive in the altered environment.

Mechanism # 6. Mismatch Repair of Single-Base Mispairs:

Many spontaneous mutations are point mutations, which involve a change in a single base pair in the DNA sequence. These can arise from errors in replication, during genetic recombination, and, particularly, by base deamination whereby a C residue is converted into a U residue. The conceptual problem with mismatch repair is determining which the normal is and which is the mutant DNA strand, and repairing the latter so that it is properly base-paired with the normal strand.

How this is accomplished has been elucidated in considerable detail for the E. coli methyl-directed mismatch repair system, often referred to as the MutHLS system. In E. coli DNA, adenine residues in a GATC sequence are methylated at the 6 position. Since DNA polymerases incorporate adenine, not methyl-adenine, into DNA, adenine residues in newly replicated DNA are methylated only on the parental strand. The adenines in GATC sequences on the daughter strands are methylated by a specific enzyme, called Dam methytransferase, only after a lag of several minutes.

During this lag period, the newly replicated DNA contains hemi-methylated GATC sequences:

An E. coli protein designated MutH, which binds specifically to hemi-methylated sequences, is able to distinguish the methylated parental strand from the un-methylated daughter strand. If an error occurs during DNA replication, resulting in a mismatched base pair near a GATC sequence, another protein, MutS, binds to this abnormally paired segment. Binding of MutS triggers binding of MufL, a linking protein that connects MutS with a nearby MutH.

This cross-linking activates a latent endonuclease activity of MutH, which then cleaves specifically the un-methylated daughter strand. Following this initial incision, the segment of the daughter strand containing the mis-incorporated base is excised and replaced with the correct DNA sequence.

E. coli strains that lack the MutS, MutH, or MutL protein have a higher rate of spontaneous mutations than wild-type cells. Strains that cannot synthesize the Dam methyltransferase also have a high rate of spontaneous mutations. Because some strains cannot methylate adenines within GATC sequences, the MwfHLS mismatch- repair system cannot distinguish between the template and newly synthesized strand and therefore cannot efficiently repair mismatched bases.

A similar mechanism repairs lesions resulting from depurination, the loss of a guanine or adenine base from DNA resulting from cleavage of the glycosidic bond between deoxyribose and the base. Depurination occurs spontaneously and is fairly common in mammals. The resulting apurinic sites, if left unrepaired, generate mutations during DNA replication because they cannot specify the appropriate paired base. All cells possess apurinic (AP) endonucleases that cut a DNA strand near an apurinic site. As with mismatch repair, the cut is extended by exonucleases, and the resulting gap then is repaired by DNA polymerase and ligase.

Mechanism # 7. Photo-reactivation UV-induced Pyrimidine Dimers:

Direct correction of damaged part can occur also in the repair of UV light induced thymine dimers. By this process of photo-reactivation or light repair, the dimers are reverted directly to the original form by exposure to visible light in the range of 320-370 nm. Photo-reactivation is catalysed by an enzyme called photolyase encoded by phr gene. When this dimer is activated by a photon of light, it splits the dimer apart. Bacterial strains with mutations in the phr gene are defective in light repair. Photolyase has been reported in prokaryotes and in lower eukaryotes but not in humans. Experimental bacteria are kept in dark to avoid photoreactivation of mutated DNA (Fig. 10.6).