© Borgis - New Medicine 1/2013, s. 14-20
*Paweł Kowalczyk1, Jolanta Krzyczkowska2, Urszula Jankiewicz3
Mechanisms of the dna repair in bacterial and yeast cells
1Autonomous Department of Microbial Biology, Faculty of Agriculture and Biology, Warsaw University of Life Sciences, Warsaw, Poland
2Department of Chemistry, Faculty of Food Sciences, Warsaw University of Life Sciences, Warsaw, Poland
3Department of Biochemistry, Warsaw University of Life Sciences, Warsaw, Poland
Malfunctioning of some DNA repair pathways predisposes to certain types of cancer. Impaired base excision repair, nucleotide excision repair, mismatch repair and recombination are implied in human tissues. These repair pathways are engaged very quickly in the cell when the external (chemical carcinogens) and internal (lipid peroxidation products) compounds react directly with DNA. This reaction may either lead to different modifications (damages) in DNA and genes. This damages may lead finally to mutation and cancer progression and induce different DNA repair mechanisms such as. BER, NER, HR and MMR. In the BER mechanisms repair is initiated by the action of a damage-specific DNA N-glycosylase that is responsible for the recognition and removal of an altered base through cleavage of the N-glycosylic bond and action of AP-endonuclease. Nucleotide excision repair (NER) is the most versatile and flexible DNA repair pathway of living cells as it deals with a wide range of structurally unrelated DNA lesions. NER corrects a wide array of DNA lesions that distort the DNA double helix, interfere in base pairing and block DNA duplication and transcription. The most common examples of these lesions are the cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4 PPs) induced by ultraviolet radiation (UV) and bases with large substitutes derived from chemicals such as polycyclic aromatic hydrocarbons or exocyclic adducts. Homologous recombination, utilize large regions of DNA homology, usually the homological chromosome, to exchange damaged DNA for the intact one. DNA mismatch-repair system (MMR) is involved in the repair of mispaired bases formed during replication, genetic recombination and as a result of DNA damage.
Different mechanisms of the DNA repair
To counteract deleterious consequences of DNA damage, the cells developed several repair mechanisms which eliminate from genomes mis-instructive or non-instructive elements, as well as seal DNA breaks. Various repair systems can be classified into the following groups, according to repair mechanism:
1. Sanitisation of nucleotide pool from modified nucleoside triphosphates, which prevents their incorporation into DNA by DNA polymerases.
2. Direct reversal of DNA damage, which occurs without breaking of the double helix.
3. Excision repair, in which a fragment of DNA strand containing the lesion is excised, followed by DNA resynthesis on the template of the opposite intact strand. Three different systems utilize the excision mode for DNA repair:
– Mismatch repair, which eliminates replication errors, some damaged DNA bases, as well as small loops.
– Base excision repair, initiated by DNA glycosylases which cleave out the damaged base and initiate the synthesis step.
– Nucleotide excision repair, in which a larger fragment of damaged DNA strand is removed (12-13 nucleotides in E. coli, 24-32 in eucaryota).
4. Recombination, which is working in the situation of gross damage and unavailability of intact opposite DNA strand or for double-strand break (DSB) repair.
5. SOS repair, based on translesion synthesis past DNA damages by specified class of low fidelity DNA polymerases tolerant of template structure abnormality, but introducing errors into DNA. Repair of exocyclic, unsubstituted etheno-DNA adducts is realized mainly by base excision repair pathway (1). 1,N2-propanoguanosine with a hexyl side chain derived from hydroxynonenal interaction with DNA was shown to be eliminated by nucleotide excision repair in mammalian cells (2, 3).
Base excision repair
Base excision repair constitutes the primary defense against lesions that do not heavily distort the DNA structure. BER is responsible for the removal of a variety of lesions. These include spontaneous hydrolytic depurination of DNA, deamination of bases, products of reaction with hydroxyl radicals, and covalent DNA adducts formed by intracellular LPO and small reactive metabolites, such as methylating agents. Repair is initiated by the action of a damage-specific DNA N-glycosylase that is responsible for the recognition and removal of an altered base through cleavage of the N-glycosylic bond (4, 5). Base removal generates an apurinic/apyrimidinic (AP) site, a noncoding DNA lesion that is both cytotoxic and mutagenic. The abasic site is the substrate for an AP endonuclease that hydrolyzes the phosphodiester bond 5” to an abasic site (6-9). Cleavage by AP-endonuclease generates a 3”-OH terminus suitable for extension by a DNA polymerase. The resulting 5” terminus contains a deoxyribose phosphate residue (dRP), which must be removed and replaced in order to complete repair. BER can proceed via two pathways, designated short patch (10) or long patch repair (11, 12). In the short patch repair pathway DNA polymerase β adds a single nucleotide and also cleaves the 5”-deoxyribose phosphate residue using an intrinsic deoxyribosephosphate lyase (dRP lyase) activity (13, 14). A DNA ligase then seals the nick to complete repair. This generates a repair product where only the damaged nucleotide is replaced. In the long patch DNA polymerase δ/ε or β and PCNA extend DNA chain at the 3”-OH terminus generated by an AP-endonuclease, displacing the strand at the 5” end. The antibodies directed against PCNA totally suppress repair patches longer than one nucleotide. This creates a baseless sugar-containing flap, removed by a human flap-endonuclease (FEN1) which is also engaged in excision of primers during replication. DNA ligase I in long patch or ligase III in the short patch seal phosphodiester bonds.
DNA glycosylases are often able to act upon a variety of DNA adducts that result from the action of a number of DNA damaging agents, although with different efficiencies and sometimes overlapping specificities. The lesions recognized by DNA glycosylases include noncanonical Watson-Crick base pairs and bases altered by deamination, oxidation and alkylation. On the basis of their primary substrate specificity all identified DNA glycosylases have been classified into several types, namely, DNA glycosylases of deaminated, mismatched, alkylated, and oxidized bases as well as of pyrimidine-dimers. DNA glycosylases are generally globular, monomeric, small proteins, ranging in molecular mass from 16 to 60 kDa. They often contain a conserved motif of helix-harpin-helix (HhH) which enables them to bind DNA. DNA glycosylases act on damaged bases by flipping damaged nucleotides out of the DNA into an enzyme active site pocket, where the excision takes place (15-17). DNA glycosylases can be separated into two groups: 1) enzymes that possess only N-glycosylase activity to generate an AP site (monofunctional DNA glycosylases), and 2) proteins that possess DNA glycosylase activity and an activity to incise the phosphodiester backbone immediately 3” of the resulting AP site via b-elimination, or 3” and 5” via b-δ-elimination, resulting in a single nucleotide gap flanked by phosphate termini (3”/5”) (bifunctional glycosylases/AP lyases) (18). Due to an associated lyase activity, bifunctional glycosylases are sometimes termed endonucleases. Cloning of the OGG1 gene from Saccharomyces cerevisiae has revealed that DNA glycosylases are not necessarily conserved throughout phylogeny, yet there is a DNA-repair protein superfamily with a wide substrate specificity found from bacteria to man.
Nucleotide excision repair (NER)
Nucleotide excision repair (NER) is the most versatile and flexible DNA repair pathway of living cells as it deals with a wide range of structurally unrelated DNA lesions. NER corrects a wide array of DNA lesions that distort the DNA double helix, interfere in base pairing and block DNA duplication and transcription. The most common examples of these lesions are the cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4 PPs), induced by ultraviolet radiation (UV), and bases with large substitutes derived from chemicals such as polycyclic aromatic hydrocarbons (19). However, NER can also correct smaller modified bases. This is the major pathway responsible for removing from DNA such exocyclic adducts as M1G and 1,N2-propanoguanine (20, 21). The overall processes of NER in eukaryotic and prokaryotic cells are similar, but there are many differences in detail. NER can be separated into two subpathways, slow, global genome repair (GGR) and fast, transcription-coupled repair (TCR). Global repair is the process by which most lesions are repaired regardless of their location in the genome. Transcription-coupled repair is characterized by the more rapid repair of lesions in the transcribed strand of an expressed gene than in the nontranscribed strand or in the rest of the genome (22). The mechanism of these two pathways is mainly similar except for recognition of the damage and, therefore, for initiation of the process. In E. coli, both subpathways require the full set of NER proteins, but transcription-coupled repair additionally requires an actively transcribing RNA polymerase (RNAP) and at least one additional factor, the transcription repair coupling factor, encoded by the mfd gene (23). The latter factor is thought to recruit Uvr proteins to RNAP arrested at a lesion on the transcribed strand (24), resulting in rapid repair of the transcription-blocking lesion.
NER in E. coli requires six proteins: UvrA, UvrB, UvrC, UvrD, DNA polymerase I, and ligase (25). In vivo, UvrA is present both as a monomer and a dimer, the latter complexing with UvrB for initial DNA damage recognition. This UvrA2B heterotrimer may carry out limited, ATP-dependent, processive scanning of the damaged region until the actual damage site is encountered (26). At this point, a conformational change occurs in the protein-DNA complex, leading to release of the UvrA dimer, stable UvrB-DNA binding, and a local bending and unwinding of the damaged region of DNA. UvrC then binds to the UvrB-DNA complex, unmasking the cryptic endonuclease activity of UvrB. In the case of UV photoproducts, this activity causes an incision to be made four bases 3” from the lesion. A second incision is made by the UvrBC complex seven bases 5” from the lesion. UvrD, also known as DNA helicase II, releases UvrC and the oligonucleotide between the dual incisions, leaving UvrB at a 12-base gap on one strand. DNA polymerase I fills the gap and dissociates the UvrB protein from the DNA. The repair process is completed by DNA ligase, which seals the nick.
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