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- 1. Chen Yonggang Zhejiang Univ. School of Medicine Research Building C-616 [email_address] 2007 A DNA Repair Overview
- 6. “ Reactive Oxygen Species” (ROS) include O•, O-O•, HOOH, •OH Thymine Thymine Glycol
- 12. An Example of Direct Repair: “Photoreactivation” MTHF or 8-HDF FADH -
- 21. Final steps of base-excision repair (replication pathway)
- 24. Early Stages of Global Genome Repair
- 25. Initial Steps of Transcription-Coupled NER
- 26. Final Steps of Eukaryotic NER
- 31. Homologous Recombination is Based on the Ability of Single DNA Strands to Find Regions of Near-Perfect Homology Elsewhere in the Genome Facilitation of Homology Searching by RecA and its Eukaryotic Homologs Eukaryotic proteins important in this process include Rad51, Rad52, Rad54, Rad55, Rad57, Rad59. BRCA1 and BRCA2 interact with Rad51 and may regulate it.
Notas do Editor
- Cancer is caused by mutations. In most cases, "genetic instability" (elevated mutation rate) is required to permit accumulation of sufficient mutations to generate cancer during a human lifetime. DNA repair mechanisms promote genomic stability and prevent cancer. Many, perhaps most, cancers are at least partially attributable to defects in DNA repair.
- Note that many types of DNA damage are generated spontaneously, in some cases at very high frequency. Thus defects in DNA repair systems are likely to cause problems even in the absence of additional damage caused by environmental factors.
- The primary amino groups of nucleic acid bases are somewhat unstable. They can be converted to keto groups in reactions like the one pictured here: In a typical mammalian cell, about 100 uracils are generated per haploid genome per day in this fashion. Other deamination reactions include conversion of adenine to hypoxanthine, guanine to xanthine, and 5-methyl cytosine to thymine.
- Chemical Modification: several types of hyper-reactive oxygen (singlet oxygen, peroxide radicals, hydrogen peroxide and hydroxyl radicals) are generated as byproducts during normal oxidative metabolism and also by ionizing radiation (X-rays, gamma rays). These are frequently called Reactive Oxygen Species (ROS). ROS can modify DNA bases. A common product of thymine oxidation is thymine glycol:
- Many environmental chemicals, including "natural" ones (frequently in the food we eat) can also modify DNA bases, frequently by addition of a methyl or other alkyl group (alkylation). In addition, normal metabolism frequently leads to alkylation. It has been shown that S-adenosylmethionine, the normal biological methyl group donor, reacts accidentally with DNA to produce alkylated bases like 3-methyladenine at a rate of several hundred per day per mammalian haploid genome. Alkylation occurs most readily at the nucleophilic positions shown in this diagram (the darker orange circles denote the most reactive positions):
- Ultraviolet light is absorbed by the nucleic acid bases, and the resulting influx of energy can induce chemical changes. The most frequent photoproducts are the consequences of bond formation between adjacent pyrimidines within one strand, and, of these, the most frequent are cyclobutane pyrimidine dimers (CPDs). T-T CPDs are formed most readily, followed by T-C or C-T; C-C dimers are least abundant. One can obtain an idea of the extent of distortion of DNA chain structure caused by CPDs by noting that, in the diagram of a T-T CPD below, the cyclobutane ring, shaded in light blue, should have sides of approximately equal length. Thus the two adjacent pyrimidines must be pulled closer to each other than in normal DNA.
- Dimers can also be produced by formation of a single covalent bond between the 6 position of one pyrimidine and the 4 position of the adjacent pyrimidine on the 3' side. The order of abundance of such pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) is T-C>>C-C>T-T>C-T. Although only one bond attaches the adjacent pyrimidines, there is nevertheless extensive distortion of the normal DNA structure. In the diagram below of the T-C 6-4PP, notice that the amino group, originally from the 4 position of the cytosine, ends up at the 5 position of the thymine. Although it is not evident from this crude diagram, the structural distortion generated by 6-4PPs (a DNA bend of 44 degrees) is significantly greater than that produced by CPDs (bend of 7-9 degrees).
- Replication errors Another major source of potential alterations in DNA is the generation of mismatches or small insertions or deletions during DNA replication. Although DNA polymerases are moderately accurate, and most of their mistakes are immediately corrected by polymerase-associated proofreading exonucleases, nevertheless the replication machinery is not perfect. As we shall see later, efficient repair mechanisms correct most of these problems. Inter-strand crosslinks By attaching to bases on both strands, bifunctional alkylating agents such as the psoralens can cross-link both strands. Cross-links can also be generated by UV and ionizing radiation. DNA-protein crosslinks DNA topoisomerases generate covalent links between themselves and their DNA substrates during the course of their enzymatic action. Usually these crosslinks are transient and are reversed as the topoisomerase action is completed. Occasionally something interferes with reversal, and a stable topoisomerase-DNA bond is established. Bifunctional alkylating agents and radiation can also create crosslinks between DNA and protein molecules. All of these lesions must be repaired. Strand breaks Single-strand and double-strand breaks are produced at low frequency during normal DNA metabolism by topoisomerases, nucleases, replication fork "collapse", and repair processes. Breaks are also produced by ionizing radiation. In fact, Hermann Müller's discovery in 1927 that X-rays can cause mutations (as a consequence of occasional failure to properly repair damage induced by ionizing radiation) was the first experimental demonstration that environmental factors can affect genome stability.
- Although it might seem that direct reversal of damage would be the simplest way to correct the damage, in most cases the reverse reaction is not possible for thermodynamic or kinetic reasons. In a few cases, the reaction is reversible, and in some of these cases mechanisms have been developed to take advantage of that reversibility. The best studied of these is photoreversal of CPDs by the enzyme, CPD photolyase. The reaction catalyzed by CPD photolyase, called "photoreactivation", was the first DNA repair process to be discovered. It was discovered by bacteriophage geneticists in 1949, well before the double-helical structure of DNA was discovered in 1953. CPD photolyase contains two chromophores responsible for absorbing light energy. In all such photolyases, one of the chromophores is FADH-, and the other is either methenyl-tetrahydrofolate (MTHF) or 8-hydroxy-5-deazaflavin (8-HDF). MTHF and 8-HDF act as primary light gatherers (colored green in the diagram below), transferring their energy to FADH- (colored yellow in the diagram). The energy from FADH- is then used to split the dimer: CPD photolyases are found in bacteria, fungi, plants and many vertebrates, but not in placental mammals. In addition, 6-4 photolyases (which repair 6-4PPs) have been found in insects, reptiles and amphibians, but not in E. coli , yeast or mammals.
- A final example of direct damage reversal is the sealing of a subset of nicks in DNA by DNA ligases. Of course, DNA ligases can only seal nicks having 5'-phosphates and 3'-hydroxyls. Nicks with other configurations, or nicks accompanied by additional backbone or base damage, require more complicated processing prior to repair and would not be classified as direct damage reversal mechanisms.
- The major DNA repair mechanisms take advantage of the facts that DNA is double-stranded and the same information is present in both strands. Consequently, in cases where damage is present in just one strand, the damage can be accurately repaired by cutting it out (excision) and replacing it with new DNA synthesized using the complementary strand as template. All organisms, prokaryotic and eukaryotic, employ at least three excision mechanisms: mismatch repair, base excision repair, and nucleotide excision repair.
- The proteins that initiate the repair process are MutS, MutL, and MutH: As implied by the above diagram, most mismatches are due to replication errors. However, mismatches can also be produced by other mechanisms--for example, by deamination of 5-methyl cytosine to produce thymidine improperly paired to G. Regardless of the mechanism by which they are produced, mismatches can always be repaired by the mismatch repair pathway. In cases where the appropriate DNA-N-glycosylase is available, mismatches can also be repaired by the base excision repair pathway (see below). The replication-error-produced mismatch in the above diagram is indicated by the distorted double helix. MutS recognizes such mismatches (true mismatches plus insertions/deletions of up to 4 nucleotides) and binds to them. Binding of MutL stabilizes the complex. E. coli DNA is normally methylated at GATC sequences, but the newly synthesized strand is not immediately methylated. The fact that the old strand, but not the new, is methylated near the replication fork allows E. coli cells to distinguish the old (presumably correct) strand from the newly-synthesized (presumably incorrect) strand. The MutS-MutL complex activates MutH, which locates a nearby methyl group and nicks the newly synthesized strand opposite the methyl group. Excision is accomplished by cooperation between the UvrD (Helicase II) protein, which unwinds from the nick toward the mismatch, and a single-strand specific exonuclease of appropriate polarity (one of several in E. coli ), followed by resynthesis (Polymerase III) and ligation (DNA ligase). It is important to note that the use of methylation to distinguish the parental strand is probably peculiar to E. coli . Data from yeast and mammalian in vitro mismatch repair experiments suggest that single-strand nicks may provide a signal for strand-specificity in these organisms. Note that single-strand breaks are present in nascent DNA strands--between Okazaki fragments in the lagging strand and at the 3' end of the leading strand. Although lacking homologs of MutH and uvrD, eukaryotic organisms possess numerous homologs of MutS and MutL:
- Specificity of the various 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: Notice that the AP site created by a DNA N-glycosylase is identical to that created by spontaneous DNA depurination or depyrimidination.
- The following diagram summarizes the initial events of BER, from introduction of base damage to action of the AP endonuclease. Uracil DNA N-glycosylase is used as an example of a glycosylase lacking AP lyase activity, and the NTH1 enzyme is used as an example of combined glycosylase and lyase activity. In the latter case, the damaged base is thymine glycol, which is represented by a T with two OH groups. Lyase activity opens the deoxyribose ring at the AP site, generating an aldehyde-terminated carbon chain. The aldehyde group is represented by an oxygen double-bonded to the carbon chain.
- In both cases above, after strand cleavage by AP endonuclease the upper strand on the left side of the nick retains a 3'-OH terminus that can easily be extended by a polymerase. In the second case, the combined action of lyase and AP endonuclease have excised the abasic deoxyribose, leaving a gap that can be easily filled in by a polymerase and sealed by a ligase. The diagram below illustrates this process using DNA polymerase beta, which is the major polymerase used for base excision repair in mammalian cells. Note that in the first case above (no lyase), the strand on the right side of the nick retains a 2'-deoxyribose-5'-phosphate terminus that must be removed to permit ligation. This non-nucleotide terminus is removed by one of two alternative pathways. In mammalian cells the major pathway is mediated by DNA polymerase beta, which has two distinct enzymatic activities. Using its polymerase activity, polymerase beta incorporates the correct nucleotide at the AP site (indicated by red bond lines and by a bold C or T), and then, with a recently discovered deoxyribose phosphatase (dRPase) activity, it excises the deoxyribose phosphate moiety, as shown below. Note that the above pathways utilizing DNA polymerase beta generate a repair "patch" (stretch of newly synthesized DNA) only a single nucleotide in length (indicated by red bond lines and bold C or T). An alternative longer patch pathway is also sometimes employed in mammalian cells and is usually employed in yeast cells.
- Although base excision repair is clearly important, it is insufficient to deal with all types of damage. For a given type of damage to be corrected by base excision repair, there must be a DNA glycosylase capable of recognizing that specific damage. The huge variety of DNA-reactive chemicals in our environment combined with the huge variety of alterations that can be produced by radiation and by oxidative and free radical attack on DNA can generate so many types of damage that coping with all types of damage by evolutionary development of damage-specific DNA glycosylases would be difficult if notimpossible. Fortunately, a different, more flexible damage repair mechanism has evolved in living organisms, nucleotide excision repair (NER) , which recognizes damaged regions based on their abnormal structure as well as on their abnormal chemistry, then excises and replaces them.
- In the above table, proteins that interact with each other sufficiently strongly to form isolatable complexes, and proteins that have similar functions, have been grouped together and provided with a common background color. Particularly noteworthy is the 10-protein complex called TFIIH (background color = green ), which is essential for DNA repair and transcription (it stimulates promoter clearing by RNA polymerase II).
- 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 HR23B proteins. The binding of XPC and HR23B initiates the process of "global genome repair" (GGR), which simply means repair anywhere in the genome. The XPC/HR23B dimer appears to recognize damaged DNA based on the extent of distortion of the normal helical DNA structure caused by the damage. Consequently, 6-4PPs are recognized more readily than CPDs. In the process of binding to the damaged region, XPC/HR23B is thought to further increase the extent of structural distortion, as illustrated in this diagram (the red box indicates a damaged site, for example a thymine dimer): The increased distortion produced by XPC/HR23B permits the entry and binding of the general transcription factor TFIIH, whose 10 subunits are colored in various shades of green in the above diagram (see also the table above). Two of these subunits (XPB and XPD; shown in brighter green) are helicases, which bind to the damaged strand and use the energy of ATP to unwind a stretch of 20-30 nucleotides including the damaged site. Three additional proteins then bind to and stabilize the open complex. The precise role of XPA is unclear, but evidence suggests that it checks to confirm that damage is present in the opened region and assists in stabilizing the open complex. RPA is the major eukaryotic single-stranded-DNA-binding protein. It is a heterotrimer, and it binds to and protects both of the separated strands in the open complex. For clarity in the diagram, it is shown binding only to the bottom strand. The long oval labeled "RPA" in the diagram represents a single RPA heterotrimer or possibly two binding side-by-side. XPG is a structure-specific nuclease (see below). Concomitant with the binding of XPA, RPA and XPG, XPC and HR23B are released. These two proteins are then free to recycle to other damaged sites where the repair process has not yet been initiated.
- Before proceeding to the next step (double strand incision), I wish to discuss another type of NER: transcription-coupled NER (TC-NER). 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. In general, the less structural distortion produced by the damage, the greater the ratio of rate of repair in transcribed strands to rate of repair elsewhere. In humans TC-NER requires all of the proteins needed for GGR except for XPC and HR23B, suggesting that a different mechanism (not requiring XPC) is involved in recognizing damage in transcribed strands. Numerous experiments suggest that this different mechanism involves the stalling of RNA polymerase at damaged sites: Defects in either of the two proteins shown associated with RNA polymerase in the above diagram, CSA and CSB, can lead to the human genetic disease, Cockayne's syndrome, which I'll discuss in more detail below. Their function is important for TC-NER, presumably in helping to recruit TFIIH to the damaged site and in helping to displace RNA polymerase and the nascent transcript so that TFIIH can access the damaged region. As in the case of GGR (above), after recruitment TFIIH unwinds a 20-30 nucleotide stretch of DNA including the damaged region. Presumably the partially unwound region produced by the stalled polymerase assists in providing access to TFIIH. The fact that the stalled polymerase produces a partially unwound region on its own may be one reason why XPC is not necessary (in humans) for TC-NER. Additional evidence, some of which is discussed below, suggests that the XPB and XPD helicase subunits of TFIIH, the TTD-A subunit of TFIIH, and also the XPG nuclease, play special roles in TC-NER--roles that go beyond their roles in GGR. It may be that these three proteins assist in the removal of RNA polymerase and RNA.
- The next step in the repair process, for both GGR and TC-NER, is recruitment of another structure-specific endonuclease, the XPF-ERCC1 heterodimer: Both XPG and XPF-ERCC1 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 on the 3' side of such a junction, while ERCC1/XPF (a heterodimeric protein complex) cuts on the 5' side. The cut made by XPG is 2-8 nucleotides from the lesion, and the cut made by ERCC1/XPF is 15-24 nucleotides away. These distances are paired with each other (probably as a consequence of the structure of the multiprotein complex) in such a way that the damage-containing oligonucleotide between the cuts averages 27 nucleotides (range 24-32 nucleotides). The damage-containing oligonucleotide is displaced concomitant with the binding of replicative gap-repair proteins (RFC, PCNA, DNA polymerase delta or epsilon), with the displacement of TFIIH, XPA, XPG, and XPF-ERCC1, and with new DNA synthesis that fills the gap. The final nick is sealed by DNA ligase I.