In this article we will discuss about the definition and mechanisms of DNA repair.
Definition of DNA Repair:
One of the main objectives of biological system is to maintain base sequences of DNA from one generation to the other. Changes in DNA sequence arise during replication of DNA damage by chemical mutagens and radiation. During replication if incorrect nucleotides have been added, they are corrected through editing system by DNA Pol I and DNA Pol III.
The other systems also exist for correcting the errors missed by editing function. It is called mismatch repair system. Mismatch repair system edits the errors left by DNA Pol I and DNA III and removes the wrong nucleotides. Proof reading by Pol I and III.
DNA is always damaged and mutated by several chemicals and radiation. Only a few errors accumulate in DNA sequence. The stable errors cause mutation and the rest are eliminated. If errors in DNA sequence are corrected before cell division, no mutation occurs. However, there are some DNA damages which cannot be mutated because the damages are not replicated. Therefore, such damages cause cell death.
There are several types of damages that occur in DNA:
(a) Modification of one or more bases by highly reactive chemicals such as alkylating agents like nitroso-amine and nitrosoguanidine,
(b) Loss of purine bases due to local pH change,
(c) Single strand or double strand break due to bending or shear forces,
(d) Dimer formation (dimerisation) between two adjacent pyrimidine molecules (e.g. T-T) due to ultraviolet and X-ray radiation.
Due to dimerisation no hydrogen bond with opposing purine shall occur. This results in distortion of helix. Most of the spontaneous errors are temporary because they are soon corrected by a process called DNA repair.
Mechanisms of DNA Repair:
There are four major pathways through which thymine-thymine dimer in DNA is repaired: light induced repair (photo-reactivation) and light-independent repair (dark repair).
The UV damages caused in cells are repaired after exposure of cells in visible light. This is called photo-reactivation. In this mechanism an enzyme DNA photolyase cleaves T-T dimer and reverse to monomeric stage (Fig. 9.17). This enzyme is activated only when exposed to visible light.
The mutant cells lack photolyase. This enzyme absorbs energy, binds of cyclobutane ring to defective sites of DNA and promotes cleavage of covalent bonds formed between T-T. This enzyme is found in several bacteria and placental mammals. Finally, thymine residues are made free and damage is repaired.
Some other photolyases catalyse DNA repair in other ways. The 6-4 photoproduct photolyase repairs the DNA damage i.e. 6-4 photoproduct caused by UV rays. The 6-4 photoproduct is formed due to formation of C4-C6 bond between two adjacent pyrimidines or due to migration of a substituent from C4 position of one pyrimidine to the C6 position of the adjoining pyrimidine. The C4 photoproduct photolyase corrects both the errors.
In Bacillus subtilis a spore photoproduct (5-thyminyl-5, 6-di-hydro-thymine) is produced after UV radiation, but not cyclobutane dimers. In light-independent reaction, photoproduct lyase is formed which repair C-C bond between the two thymines.
(ii) Excision Repair:
It is an enzymatic process. In this mechanism, the damaged portion is removed and replaced by new DNA. The second DNA strand acts as template for the synthesis of new DNA fragment.
Excision repair involves DNA of different lengths such as:
(a) Very short patch repair
(b) Short patch repair, and
(c) Long patch repair.
The very short patch repair includes the mismatch of a single base, while the latter two deals with mismatches in a long patches of the DNA. The short and long patches of damaged DNA molecules are repaired by uvr genes for example uvr A, B C and D which encode repair endonuclease.
(a) Base excision repair:
The lesions containing non-helix distortion (e.g. alkylating bases) are repaired by base excision repair. It involves at least six enzymes called DNA glycosylases.
Each enzyme recognises at least bases and removes from DNA strand. The enzymes remove deaminated cytosine, deaminated adenine, alkylated or oxidised base. Base excision repair pathway starts with a DNA glycosylation. For example, the enzyme uracyl DNA glycosylase removes the uracyl that has wrongly joined with G which is really deaminated cytosine (Fig. 9.18A).
Then AP- endonuclease (apurinic or apyriminic site) and phosphodiesterase removes sugar-phosphate. AP- sites arise as a result of loss of a purine or a pyrimidine. A gap of single nucleotide develops on DNA which acts as template-primer for DNA polymerase to synthesise DNA and fill the gap by DNA lygase.
(b) Nucleotide excision repair:
Any type of damage having a large change in DNA helix causing helical changes in DNA structure is repaired by this pathway. Such damage may arise due to pyrimidine dimers (T-T, T-C and C-C) caused by sun light and covalently joins large hydrocarbon (e.g. the carcinogen benzopyrene).
In E. coli a repair endonuclease recognises the distortion produced by T-T dimer and makes two cuts in the sugar phosphate backbone on each side of the damage. The enzyme DNA helicases removes oligonucleotide from the double helix containing damage. DNA polymerase III and DNA ligase repair the gap produced in DNA helix (Fig. 9.18B).
(c) Recombination repair (daughter-strand gap repair):
When excision repair mechanisms fails, this mechanism, is required to repair errors. This mechanisms, operates in the viral chromosome in host cell whose DNA is damaged. This mechanism operates only after replication; therefore, it is also known as post-replication repair.
Probably RecA protein in E. coli catalyses DNA strand for sister-strand exchange. Thus a single stranded DNA segment without any defect is excised from a strand on the homologous DNA segment at the replication fork.
It is inserted into the gap created by excision of thymine dimer (Fig. 9.19). Then the combined action of DNA Pol I and DNA ligase joins the inserted piece. The gap formed in donor DNA molecule is also filled by DNA Pol I and ligase enzymes.
(d) Methylation-directed very short patch repair:
Very short patch (VSP) repair is accomplished by involving methylation of bases especially cytosine and adenine. In E. coli methylation of adenine and in a sequence of -GATC- is done by the enzyme methylase (a product of dam gene) on both strands of DNA. After replication only A of -GATC- of one strand remains methylated, while the other remains un-methylated until methylase accomplishes methylation (Fig. 9.20 A-B).
In E. coli repairing activity required four proteins viz., Mutl, MutS, MutU (UvrS) and MutH by the genes mutL, mutS, mutU, and mutH, respectively. The mut genes are the loci which increase the frequency of spontaneous mutation. The un-methylated -GATC allows the MutL to recognise the mismatch during transition period.
This helps MutS to bind to mismatch. MutU supports in unwinding the single strand and single strand DNA binding (SSB) proteins and maintain the structural topography of single strand. MutH cleaves the newly synthesised DNA strand and the protein MutU separates the mismatch strand (A).
However, there is a gradient of methylation along the newly synthesised strand. Least methylation occurs at the replication fork. The parental strand is uniformly methylated.
The methylated bases direct the excision mechanisms to the newly synthesised strand containing the incorrect nucleotides (B). During this transition period, the repair system works and distinguishes the old and new strands and repairs only the new strands.
(e) SOS Repair:
SOS (Save Our Soul) repair is a by-pass repair system. It is also called emergency repair. The damage in DNA itself induces the, SOS regulatory system which is a complex cellular mechanism.
SOS works where photo-dimers are formed that lead to cell death. SOS is the last attempt to minimise mutation for survival. It induces a number of DNA repair processes. SOS system works in the absence of a DNA template. Therefore, many errors arise leading to mutation.
Generally genes of SOS system remains in repressed condition caused by a protein LexA. Repression of SOS is inactivated by RecA protease. It is formed after the conversion of RecA protein by DNA damage.
DNA damage results in conversion of RecA protein into RecA protease (Fig. 9.21A). RecA protease breaks LexA protein (B). Normally, LexA protein inhibits the activity or recA gene (C) and the DNA repair genes (uvrA and umuD) (D).
Finally, DNA repair genes are activated. SOS system does not repair the large amount of damage. When DNA repair is over, RecA protein loses its proteolytic activity. Then LexA protein accumulates and binds to SOS operator and turns off SOS operator (D). However, repression is not complete. Beside, some RecA protein is also produced that inactivates LexA protein.