DNA repair - BER and NER mechanisms
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DNA repair – BER and NER mechanisms DNA Repair Cells possess a large number of different types of repair systems. Those repair systems can be grouped into main several broad categories: •Direct reversal of damage – as the name suggests, these systems act directly on damaged nucleotides, converting each one back to its original structure. •Excision of damaged region, followed by precise replacement: Base excision repair Nucleotide excision repair Mismatch repair •Recombination repair is used to mend double-strand breaks •Damage tolerance – tries to minimize the effects of damage that has not been repaired. 1
Direct repair systems Direct repair systems fill in nicks and correct some types of nucleotide modification Relatively few forms of DNA damage can be repaired without excision of nucleotides. Those that can be repaired by direct methods are as follows: Nicks Nicks can be repaired by a ligase if just a phosphodiester bond has been broken, without damage to the 5’ phosphate and 3’ hydroxyl groups of the nucleotides at either side of the nick. Nicks with other configurations, or nicks accompanied by additional backbone or base damage, require more complicated excision repair mechanisms. Alkylation enzymes Some forms of alkylation damage are directly reversible by special enzymes that transfer alkyl groups from the nucleotide to their peptide chains. Enzymes capable of doing this are known in many organisms. These include the Ada enzyme of E. coli, which is involved in adaptive process that this bacterium is able to activate in response to DNA damage. Ada removes alkyl groups attached to oxygen groups at positions 4 and 6 of thymine and guanine, respectively. 2
Alkyl transfer in eukaryotes •Direct repair mechanism. •Enzymic transfer of methyl group from O6-MeG to residue in methyl transferase (MGMT) •O6-MeG is cytotoxic, mutagenic and tumorogenic. •20% of human tumour cell lines are MGMT deficient and MGMT may have a significant role in cancer prevention. •No known disease associated with mutation in MGMT gene. 3
Removal of CPDs – light repair Photoreactivaton •Direct repair mechanism, evidence for existence of photoreactivation in human cells is controversial. •Enzymic reversal of PP dimers (caused by UV light and a major cause of skin cancer) to monomers. Cyclobutyl dimers are repaired by a light-dependent direct system called photoreactivation. This process is done by a special enzyme CPD photolyase, that does photo-reversal of CPDs. CPD photolyases are found in bacteria, fungi, plants and many vertebrates, but not in mammals. In addition, there are 6-4 photolyases that repair 6-4PPs. Those were found in insects, reptiles and amphibians, but not in E. coli, yeast or mammals. CPD photolyases 4
Base excision repair (BER) Base excision repair: •Repair of small, non-bulky DNA lesions (methylated, oxidised, reduced bases) •Modified or damaged base is removed by a DNA glycosylase (several glycosylases have been described, including uracil-DNA glycosylase), creating an apurinic or apyrimidinic (AP) site. •AP-deoxyribose is then released by AP exonucleases. Missing nucleotide replaced by DNA polymerase and ligated. •No known human diseases associated with defects in base excision. 5
Base Excision Repair (BER) Base excision repair is the least complex of the various repair systems. It is used to repair modified nucleotides that have suffered relatively minor damage. Done by special DNA glycosylases. Eg. It can remove incorrect bases (like uracil) or damaged bases (like 3-methyladenine). 3 main steps: 1. Removal of the incorrect base by an appropriate DNA N- glycosylase to create an AP site. AP site is identical to one created by spontaneous base loss. 2. Nicking of the damaged DNA strand by AP endonuclease upstream of the AP site, thus creating a 3'-OH terminus adjacent to the AP site 3. Extension of the 3'-OH terminus by a DNA polymerase, accompanied by excision of the AP site 6
Base Excision Repair (BER) Specificity of the various BER pathways is conferred by the DNA N- glycoslyases. These hydrolyze the N-glycosylic bond between the base and the deoxyribose, as illustrated here by the action of uracil DNA N- glycosylase (Scheme by Dr. Huberman) DNA Glycosylases •Uracil DNA N-glycosylase; •Thymine DNA glycosylase, •Methyl Purine DNA glycosylase; •8-Oxo-Guanine glycolyase 1; •Endonuclease Three Homolog 1 (NTH1) (does T-glycol, formamidopyrimidine…) 7
BER A battery of glycosylases, each dealing with a relatively narrow, partially overlapping spectrum of lesions, feeds into a core reaction. Glycosylases flip the suspected base out of the helix by DNA backbone compression to accommodate it in an internal cavity of the protein. Inside the protein, the damaged base is cleaved from the sugar-phosphate backbone (stage I in the figure). BER The resulting abasic site can also occur spontaneously by hydrolysis. The core BER reaction is initiated by strand incision at the abasic site by the APE1 endonuclease (II). 8
BER Poly(ADP-ribose) polymerase (PARP), which binds to and is activated by DNA strand breaks, and the recently identified polynucleotide kinase (PNK) may be important when BER is initiated from a SSB to protect and trim the ends for repair synthesis (III). BER In mammals, the so- called short-patch repair is the dominant mode for the remainder of the reaction. DNA pol performs a one- nucleotide gap-filling reaction (IV) and removes the 5'-terminal baseless sugar residue via its lyase activity (V); this is then followed by sealing of the remaining nick by the XRCC1–ligase3 complex (VI). 9
BER The XRCC1 scaffold protein interacts with most of the above BER core components and may therefore be instrumental in protein exchange. The long-patch repair mode involves DNA pol, and proliferating cell nuclear antigen (PCNA) for repair synthesis (2–10 bases) as well as the FEN1 endonuclease to remove the displaced DNA flap and DNA ligase 1 for sealing (VII–IX). BER The above BER reaction operates across the genome. However, some BER lesions block transcription, and in this case the problem is dealt with by the TCR pathway described above, including TFIIH, XPG (which also stimulates some of the glycosylases) and probably the remainder of the core NER apparatus. 10
References Hoeijmakers, J. Genome maintenance mechanisms for preventing cancer. Nature 411, 366-374 (2001). J. Huberman (2001) DNA repair. Roswell Park Cancer Institute. T. A. Brown, Genomes, 1999, Wiley-Liss, New-York. R. Weaver, Molecular biology, 2003. Nucleotide excision repair (NER). •Sole repair system for bulky DNA lesions, also repairs smaller types of lesion: no known covalent base modification which is not a substrate for this system. •There are two modes of NER in eukaryotes: global-genome NER and transcription-coupled NER. •Xeroderma pigmentosum is associated with defects common to both NER pathways. •Defects in TC-NER are associated with Cockayne syndrome. 11
General steps of NER: 1. Damage recognition 2. Binding of a multi-protein complex at the damaged site 3. Double incision of the damaged strand several nucleotides away from the damaged site, on both the 5' and 3' sides 4. Removal of the damage-containing oligonucleotide from between the two nicks 5. Filling in of the resulting gap by a DNA polymerase 6. Ligation Nucleotide excision repair (NER) Best studies example is the short patch process in E.coli, the region replaces is usually 12 nt in length. Short patch repair is initiated by multienzyme complex UvrABC system. 12
Substrates for the UvrABC endonuclease of E. coli. Substrates for the UvrABC endonuclease of E. coli. 13
Excision repair of DNA by E. coli UvrABC mechanism Two molecules of UvrA and one of UvrB form the complex that moves randomly along DNA. Once a complex finds a lesion, conformational changes in DNA, powered by ATP hydrolysis, cause the helix to become locally denatured and kinked by 130o . After UvrA dimer dissociates, UvrC endonuclease binds next to the UvrB protein UvrC activates the UvrB protein to nick the DNA approximately 4 nucleotides 3' to the damaged site. This activates UvrC to nick the DNA approximately 7 nucleotides 5' to the damage. It is possible that activation of UvrC is a consequence of a conformational change in the DNA after nicking by UvrB. These steps all require ATP binding but not ATP hydrolysis UvrD helicase action A helicase, (UvrD) uses the energy of ATP, unwinds damaged region, releasing single stranded fragment with the lesion, which is degraded to mononucleotides. UvrC and Uvr B are displaced. The gap is filled by DNA Polymerase I, and the remaining nick is sealed by DNA ligase. 14
NER in eukaryotic cells 15
NER in eukaryotic cells The initial steps depend on whether the damage is in the actively transcribed strand of a gene or elsewhere in the genome. If the damage is not in the actively transcribed strand of a gene, then the damage is recognized and bound by a heterodimer consisting of the XPC and hHR23B proteins. The binding of XPC and hHR23B initiates the process of "global genome repair" (GGR), which simply means repair anywhere in the genome. The XPC/hHR23B dimer appears to recognize damaged DNA based on the extent of distortion of the normal helical DNA structure caused by the damage. In the process of binding to the damaged region, XPC/hHR23B is thought to further increase the extent of structural distortion. Scheme of Dr. Huberman NER in eukaryotic cells Scheme by Dr. Huberman The increased distortion produced by XPC/hHR23B permits the entry and binding of three additional proteins or protein complexes:TFIIH, whose 9 subunits (green shades) . Two of these subunits (XPB and XPD; shown in brighter green) are helicases, which bind to the damaged strand and cooperate in unwinding in opposite directions and with RPA (the eukaryotic single-stranded DNA binding protein complex) and XPA to generate an unwound stretch of 20-30 nucleotides including the damaged site. XPA is essential for complete unwinding and for NER, but its precise role is still unclear. Because XPA binds preferentially to damaged DNA on its own and also interacts with TFIIH and RPA, it is likely to cooperate with XPC/hHR23B in recruiting TFIIH and RPA to the damaged region. It may also help to position the other proteins properly with respect to the damaged site. Next step is double strand incision. 16
Another type of NER: transcription-coupled repair (TCR) - within transcribed strand. NB: Numerous experiments have demonstrated that damage within the transcribed strands of genes is usually repaired more rapidly than damage in the non-transcribed strand or damage in non-gene regions. NB: the less structural distortion produced by the damage, the greater the ratio of rate of repair in transcribed strands to rate of repair elsewhere. TCR requires all of the proteins needed for GGR except for XPC, suggesting that a different mechanism (not requiring XPC) is involved in recognizing damage in transcribed strands. This mechanism involves the stalling of RNA polymerase at damaged sites: Scheme by Dr. Huberman The two proteins shown associated with RNA polymerase are CSA and CSB. Those were found defective in the human genetic disease, Cockayne's syndrome. Their function is important for TCR, presumably in helping to recruit TFIIH, XPA and RPA to the damaged site. They also help to displace RNA polymerase sufficiently so that TFIIH, XPA and RPA can access the damaged region. Similarly with GGR, after recruitment these three proteins/protein complexes unwind a 20-30 nucleotide stretch of DNA near the damaged region. Presumably the partially unwound region produced by the stalled polymerase helps in providing access to TFIIH, XPA and RPA. The fact that the stalled polymerase produces a partially unwound region on its own may be one reason why XPC is not necessary for TCR. The efficiency of TCR is undoubtedly also enhanced by the fact that TFIIH is a transcription initiation factor and is therefore likely to interact with stalled RNA polymerases (scheme by Dr. Huberman) 17
Final step – recruitment of nucleases The next step in the repair process, for both GGR and TCR, is recruitment of two structure-specific endonucleases, XPG and XPF/ERCC1 Final step – recruitment of nucleases Both nucleases are specific for junctions between single- and double- stranded DNA. XPG, which is closely related to the FEN-1 nuclease that participates in base excision repair, cuts within the dsDNA on the 3' side of such a junction. ERCC1/XPF (a heterodimeric protein complex) cuts on the 5' side. Biochemical studies suggest that the incision by XPG precedes the incision by ERCC1/XPF. The cut made by XPG is 2-8 nucleotides from the lesion, and the cut made by ERCC1/XPF is 15-24 nucleotides away – this all together results on cuts averages 27 nucleotides (range 24-32 nucleotides). 18
The mechanism by which the damage-containing oligonucleotide is displaced is not clear. Perhaps the XPB/XPD helicases assist in this function. After the oligonucleotide is removed, the resulting gap is filled in by DNA polymerase epsilon or delta, together with PCNA The final nick is sealed by DNA ligase I. Model for mechanism of global genome NER and TCR The GG-NER-specific complex XPC- hHR23B screens first on the basis of disrupted base pairing, instead of lesions per se. This explains why mildly distorting injury such as cyclobutane pyrimidine dimers are poorly repaired. 19
Model for mechanism of global genome NER and TCR In TCR, the ability of a lesion (whether of the NER- or BER-type) to block RNA polymerase seems critical (stage I in the figure opposite). The stalled polymerase must be displaced to make the injury accessible for repair, and this requires at least two TCR-specific factors: CSB and CSA. Model for mechanism of global genome NER and TCR The subsequent stages of GG-NER and TCR may be identical. The XPB and XPD helicases of the multi- subunit transcription factor TFIIH open 30 base pairs of DNA around the damage (II). XPA probably confirms the presence of damage by probing for abnormal backbone structure, and when absent aborts NER. 20
Model for mechanism of global genome NER and TCR The single-stranded-binding protein RPA (replication protein A) stabilizes the open intermediate by binding to the undamaged strand (III). The use of subsequent factors, each with limited capacity for lesion detection in toto, still allows very high damage specificity. Model for mechanism of global genome NER and TCR The endonuclease duo of the NER team, XPG and ERCC1/XPF, respectively cleave 3' and 5' of the borders of the opened stretch only in the damaged strand, generating a 24–32-base oligonucleotide containing the injury (IV). 21
Model for mechanism of global genome NER and TCR The regular DNA replication machinery then completes the repair by filling the gap (V). In total, 25 or more proteins participate in NER. In vivo studies indicate that the NER machinery is assembled in a step-wise fashion from individual components at the site of a lesion. After a single repair event (which takes several minutes) the entire complex is disassembled again. 22
Literature sources: T.A. Brown. Genomes, John Wiley and Sons,Inc., New- York,p. 330-350 (1999). E.Friedberg, G. Walker, W. Siede. DNA repair and mutagenesis, ASM press, Washington DC, 1995 B. Lewin. Genes VII, Oxford University Press. J. Huberman (2001) DNA repair. Roswell Park Cancer Institute. R. Weaver, Molecular Biology, 2003, McGraw Hill Hoeijmakers, J. Genome maintenance mechanisms for preventing cancer. Nature 411, 366-374 (2001). Literature sources: T.A. Brown. Genomes, John Wiley and Sons,Inc., New- York,p. 330-350 (1999). E.Friedberg, G. Walker, W. Siede. DNA repair and mutagenesis, ASM press, Washington DC, 1995 B. Lewin. Genes VII. J. Huberman (2001) DNA repair. Roswell Park Cancer Institute. R. Weaver, Molecular Biology, 2003, McGraw Hill 23
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