The second part of this chapter examines the major classes of DNA repair processes. These are:
- reversal of damage,
- nucleotide excision repair,
- base excision repair,
- mismatch repair,
- recombinational repair, and
- error-prone repair.
Many of these processes were first studies in bacteria such as E. coli, however only a few are limited to this species. For instance, nucleotide excision repair and base excision repair are found in virtually all organisms, and they have been well characterized in bacteria, yeast, and mammals. Like DNA replication itself, repair of damage and misincorporation is a very old process.
Reversal of damage
Some kinds of covalent alteration to bases in DNA can be directly reversed. This occurs by specific enzyme systems recognizing the altered base and breaking bonds to remove the adduct or change the base back to its normal structure.
Photoreactivation is a light-dependent process used by bacteria to reverse pyrimidine dimers formed by UV radiation. The enzyme photolyase binds to a pyrimidine dimer and catalyzes a second photochemical reaction (this time using visible light) that breaks the cyclobutane ring and reforms the two adjacent thymidylates in DNA. Note that this is not formally the reverse of the reaction that formed the pyrimidine dimers, since energy from visible light is used to break the bonds between the pyrimidines, and no UV radiation is released. However, the result is that the DNA structure has been returned to its state prior to damage by UV. The photolyase enzyme has two subunits, which are encoded by the phrA and phrBgenes in E. coli.
A second example of the reversal of damage is the removal of methyl groups. For instance, the enzyme O6‑methylguanine methyltransferase, encoded by the adagene in E. coli, recognizes O6‑methylguanine in duplex DNA. It then removes the methyl group, transferring it to an amino acid of the enzyme. The methylated enzyme is no longer active, hence this has been referred to as a suicide mechanism for the enzyme.
The most common means of repairing damage or a mismatch is to cut it out of the duplex DNA and recopy the remaining complementary strand of DNA, as outlined in Fig. 7.12. Three different types of excision repair have been characterized: nucleotide excision repair, base excision repair, and mismatch repair. All utilize a cut, copy, and paste mechanism. In the cuttingstage, an enzyme or complex removes a damaged base or a string of nucleotides from the DNA. For the copying, a DNA polymerase (DNA polymerase I in E. coli) will copy the template to replace the excised, damaged strand. The DNA polymerase can initiate synthesis from 3' OH at the single-strand break (nick) or gap in the DNA remaining at the site of damage after excision. Finally, in the pastingstage, DNA ligase seals the remaining nick to give an intact, repaired DNA.
Figure 7.12. A general scheme for excision repair, illustrating the cut (steps 1 and 2), copy (step 3) and paste (step 4) mechanism.
Nucleotide Excision Repair (NER)
In nucleotide excision repair, damaged bases are cut out within a string of nucleotides, and replaced with DNA as directed by the undamaged template strand. This repair system is used to remove pyrimidine dimers formed by UV radiation as well as nucleotides modified by bulky chemical adducts. The common feature of damage that is repaired by nucleotide excision is that the modified nucleotides cause a significant distortion in the DNA helix. NER occurs in almost all organisms examined.
Some of the best-characterized enzymes catalyzing this process are the UvrABC excinuclease and the UvrD helicase in E. coli. The genes encoding this repair function were discovered as mutants that are highly sensitive to UV damage, indicating that the mutants are defective in UV repair. As illustrated in Fig. 7.13, wild type E. coli cells are killed only at higher doses of UV radiation. Mutant strains can be identified that are substantially more sensitive to UV radiation; these are defective in the functions needed for UV-resistance, abbreviated uvr. By collecting large numbers of such mutants and testing them for their ability to restore resistance to UV radiation in combination, complementation groups were identified. Four of the complementation groups, or genes, encode proteins that play major rules in NER; they are uvrA, uvrB, uvrCand uvrD.
Figure 7.13. Survival curve of bacteria exposed to UV radiation. Cultures of bacteria are exposed to increasing doses of UV radiation, plotted along the horizontal axis. Samples of each irradiated culture are then plated and the number of surviving colonies are counted (plotted as a logarithmic function on the vertical axis). Mutant strains that are more sensitive to UV damage are defective in the genes that confer UV-resistance, i.e. they are defective in uvr functions.
The enzymes encoded by the uvrgenes have been studied in detail. The polypeptide products of the uvrA, uvrB, and uvrCgenes are subunits of a multisubunit enzyme called the UvrABC excinuclease. UvrA is the protein encoded by uvrA, UvrB is encoded by uvrB, and so on. The UvrABC complex recognizes damage-induced structural distortions in the DNA, such as pyrimidine dimers. It then cleaves on both sides of the damage. Then UvrD (also called helicase II), the product of the uvrDgene, unwinds the DNA, releasing the damaged segment. Thus for this system, the UvrABC and UvrD proteins carry out a series of steps in the cutting phase of excision repair. This leaves a gapped substrate for copying by DNA polymerase and pasting by DNA ligase.
The UvrABC proteins form a dynamic complex that recognizes damage and makes endonucleolytic cuts on both sides. The two cuts around the damage allow the single-stranded segment containing the damage to be excised by the helicase activity of UvrD. Thus the UvrABC dynamic complex and the UvrBC complex can be called excinucleases. After the damaged segment has been excised, a gap of 12 to 13 nucleotides remains in the DNA. This can be filled in by DNA polymerase and the remaining nick sealed by DNA ligase. Since the undamaged template directs the synthesis by DNA polymerase, the resulting duplex DNA is no longer damaged.
In more detail, the process goes as follows (Fig. 7.14). UvrA2 (a dimer) and Uvr B recognize the damaged site as a (UvrA)2UvrB complex. UvrA2 then dissociates, in a step that requires ATP hydrolysis. This is an autocatalytic reaction, since it is catalyzed by UvrA, which is itself an ATPase. After UvrA has dissociated, UvrB (at the damaged site) forms a complex with UvrC. The UvrBC complex is the active nuclease. It makes the incisions on each side of the damage, in another step that requires ATP. The phosphodiester backbone is cleaved 8 nucleotides to the 5' side of the damage and 4-5 nucleotides on the 3' side. Finally, the UvrD helicase then unwinds DNA so the damaged segment is removed. The damaged DNA segment dissociates attached to the UvrBC complex. Like all helicase reactions, the unwinding requires ATP hydrolysis to disrupt the base pairs. Thus ATP hydrolysis is required at three steps of this series of reactions.
Figure 7.14.The Uvr(A)BC excinuclease of E. coli recognizes AP sites, thymine dimers, and other structural distortions and makes nicks on both sides of the damaged region. The 12-13 nucleotide-long fragment is released together with the excinuclease by helicase II action.
How does an excinuclease differ from an exonuclease and an endonuclease?
Nucleotide excision repair is very active in mammalian cells, as well as cells from may other organisms. The DNA of a normal skin cell exposed to sunlight would accumulate thousands of dimers per day if this repair process did not remove them! One human genetic disease, called xeroderma pigmentosum (XP), is a skin disease caused by defect in enzymes that remove UV lesions. Fibroblasts isolated from individual XP patients are markedly sensitive to UV radiation when grown in culture, similar to the phenotype shown by E. coliuvrmutants. These XP cell lines can be fused in culture and tested for the ability to restore resistance to UV damage. XP cells lines that do so fall into different complementation groups. Several complementation groups, or genes, have been defined in this way. Considerable progress has been made recently in identifying the proteins encoded by each XP gene (Table 7.2). Note the tight analogy to bacterial functions needed for NER. Similar functions are also found in yeast (Table 7.2). Additional proteins utilized in eukaryotic NER include hHR23B (which forms a complex with the DNA-damage sensor XPC), ERCCI (which forms a complex with the XPF to catalyze incision 5’ to the site of damage), the several other subunits of TFIIH (see Chapter 10) and the single-strand binding protein RPA.
|Human Gene||Protein Function||Homologous to S. cerevisiae||Analogous to E. coli|
|XPA||Binds damaged DNA||Rad14||UvrA/UvrB|
|XPB||3’ to 5’ helicase, component of TFIIH||Rad25||UvrD|
|XPC||DNA-damage sensor (in complex with hHR23B)||Rad4|
|XPD||5’ to 3’ helicase, component of TFIIH||Rad3||UvrD|
|XPE||Binds damaged DNA||UvrA/UvrB|
|XPF||Works with ERRC1 to cut DNA on 5’ side of damage||Rad1||UvrB/UvrC|
|XPG||Cuts DNA on 3’ side of damage||Rad2||UvrB/UvrC|
NER occurs in two modes in many organisms, including bacteria, yeast and mammals. One is the global repair that acts throughout the genome, and the second is a specialized activity is that is coupled to transcription. Most of the XP gene products listed in Table 2 function in both modes of NER in mammalian cells. However, XPC (acting in a complex with another protein called hHR23B) is a DNA-damage sensor that is specific for global genome NER. In transcription coupled NER, the elongating RNA polymerase stalls at a lesion on the template strand; perhaps this is the damage recognition activity for this mode of NER. One of the basal transcription factors that associates with RNA polymerase II, TFIIH (see Chapter 10), also plays a role in both types of NER. A rare genetic disorder in humans, Cockayne syndrome (CS), is associated with a defect specific to transcription coupled repair. Two complementation groups have been identified, CSAand CSB. Determination of the nature and activity of the proteins encoded by them will provide additional insight into the efficient repair of transcribed DNA strands. The phenotype of CS patients is pleiotropic, showing both photosensitivity and severe neurological and other developmental disorders, including premature aging. These symptoms are more severe than those seen for XP patients with no detectable NER, indicating that transcription-coupled repair or the CS proteins have functions in addition to those for NER.
Other genetic diseases also result from a deficiency in a DNA repair function, such as Bloom's syndrome and Fanconi's anemia. These are intensive areas of current research. A good resource for updated information on these and other inherited diseases, as well as human genes in general, is the Online Mendelian Inheritance in Man, or OMIM, accessible at http://www.ncbi.nlm.nih.gov.
Ataxia telangiectasia, or AT, illustrates the effect of alterations in a protein not directly involved in repair, but perhaps signaling that is necessary for proper repair of DNA. AT is a recessive, rare genetic disease marked by uneven gait (ataxia), dilation of blood vessels (telangiectasia) in the eyes and face, cerebellar degeneration, progressive mental retardation, immune deficiencies, premature aging and about a 100-fold increase in susceptibility to cancers. That latter phenotype is driving much of the interest in this locus, since heterozygotes, which comprise about 1% of the population, also have an increased risk of cancer, and may account for as much as 9% of breast cancers in the United States. The gene that is mutated in AT (hence called "ATM") was isolated in 1995 and localized to chromosome 11q22-23.
The ATM gene does not appear to encode a protein that participates directly in DNA repair (unlike the genes that cause XP upon mutation). Rather, AT is caused by a defect in a cellular signaling pathway. Based on homologies to other proteins, the ATM gene product may be involved in the regulation of telomere length and cell cycle progression. The C-terminal domain is homologous to phosphatidylinositol-3-kinase (which is also a Ser/Thr protein kinase) - hence the connection to signaling pathways. The ATM protein also has regions of homology to DNA-dependent protein kinases, which require breaks, nicks or gaps to bind DNA (via subunit Ku); binding to DNA is required for the protein kinase activity. This suggests that ATM protein could be involved in targeting the repair machinery to such damage.
Base Excision Repair
Base excision repair differs from nucleotide excision repair in the types substrates recognized and in the initial cleavage event. Unlike NER, the base excision machinery recognizes damaged bases that do not cause a significant distortion to the DNA helix, such as the products of oxidizing agents. For example, base excision can remove uridines from DNA, even though a G:U base pair does not distort the DNA. Base excision repair is versatile, and this process also can remove some damaged bases that do distort the DNA, such as methylated purines. In general, the initial recognition is a specific damaged base, not a helical distortion in the DNA. A second major difference is that the initial cleavage is directed at the glycosidic bond connecting the purine or pyrimidine base to a deoxyribose in DNA. This contrasts with the initial cleavage of a phosphodiester bond in NER.
Cells contain a large number of specific glycosylases that recognize damaged or inappropriate bases, such as uracil, from the DNA. The glycosylase removes the damaged or inappropriate base by catalyzing cleavage of the N-glycosidic bond that attaches the base to the sugar-phosphate backbone. For instance, uracil-N-glycosylase, the product of the ung gene, recognizes uracil in DNA and cuts the N-glycosidic bond between the base and deoxyribose (Fig. 7.15). Other glycosylases recognize and cleave damaged bases. For instance methylpurine glycosylase removes methylated G and A from DNA. The result of the activity of these glycosylases is an apurinic/apyrimidinic site, or AP site (Fig. 7.15). At an AP site, the DNA is still an intact duplex, i.e. there are no breaks in the phosphodiester backbone, but one base is gone.
Next, an AP endonuclease nicks the DNA just 5’ to the AP site, thereby providing a primer for DNA polymerase. In E. coli, the 5' to 3' exonuclease function of DNA polymerase I removes the damaged region, and fills in with correct DNA (using the 5' to 3' polymerase, directed by the sequence of the undamaged complementary strand).
Additional mechanisms have evolved for keeping U’s out of DNA. E. colialso has a dUTPase, encoded by the dutgene, which catalyzes the hydrolysis of dUTP to dUMP. The product dUMP is the substrate for thymidylate synthetase, which catalyzes conversion of dUMP to dTMP. This keeps the concentration of dUTP in the cell low, reducing the chance that it will be used in DNA synthesis. Thus the combined action of the products of the dut+ unggenes helps prevent the accumulation of U's in DNA.
In base excision repair, which enzymes are specific for a particular kind of damage and which are used for all repair by this pathway?
Figure 7.15. Base excision repair is initiated by a glycosylase that recognizes and removes chemically damaged or inappropriate bases in DNA. The glycosylase cleaves the glycosidic bond between the base and the sugar, leaving an apurinic/apyrimidinic site. The AP endonuclease can then nick the phosphodiester backbone 5’ to the AP site. When DNA polymerase I binds the free primer end at the nick, its 5'-3' exonuclease activity cuts a few nucleotides ahead of the missing base, and its polymerization activity fills the entire gap of several nucleotides.
The third type of excision repair we will consider is mismatch repair, which is used to repair errors that occur during DNA synthesis. Proofreading during replication is good but not perfect. Even with a functional e subunit, DNA polymerase III allows the wrong nucleotide to be incorporated about once in every 108 bp synthesized in E. coli. However, the measured mutation rate in bacteria is as low as one mistake per 1010 or 1011 bp. The enzymes that catalyze mismatch repairare responsible for this final degree of accuracy. They recognize misincorporated nucleotides, excise them and replace them with the correct nucleotides. In contrast to nucleotide excision repair, mismatch repair does not operate on bulky adducts or major distortions to the DNA helix. Most of the mismatches are substitutes within a chemical class, e.g. a C incorporated instead of a T. This causes only a subtle helical distortions in the DNA, and the misincorporated nucleotide is a normal component of DNA. The ability of a cell to recognize a mismatch reflects the exquisite specificity of MutS, which can distinguish normal base pairs from those resulting from misincorporation. Of course, the repair machinery needs to know which of the nucleotides at a mismatch pair is the correct one and which was misincorporated. It does this by determining which strand was more recently synthesized, and repairing the mismatch on the nascent strand.
In E. coli, the methylation of A in a GATC motif provides a covalent marker for the parental strand, thus methylation of DNA is used to discriminate parental from progeny strands. Recall that the dam methylase catalyzes the transfer of a methyl group to the A of the pseudopalindromic sequence GATC in duplex DNA. Methylation is delayed for several minutes after replication. IN this interval before methylation of the new DNA strand, the mismatch repair system can find mismatches and direct its repair activity to nucleotides on the unmethylated, newly replicated strand. Thus replication errors are removed preferentially.
The enzyme complex MutH-MutL-MutS , or MutHLS, catalyzes mismatch repair in E. coli. The genes that encode these enzymes, mutH, mutLand mutS, were discovered because strains carrying mutations in them have a high frequency of new mutations. This is called a mutator phenotype, and hence the name mutwas given to these genes. Not all mutator genes are involved in mismatch repair; e.g., mutations in the gene encoding the proofreading enzyme of DNA polymerase III also have a mutator phenotype. This gene was independently discovered in screens for defects in DNA replication (dnaQ ) and mutator genes (mutD). Three complementation groups within the set of mutator alleles have been implicated primarily in mismatch repair; these are mutH, mutLand mutS.
MutS will recognize seven of the eight possible mismatched base pairs (except for C:C) and bind at that site in the duplex DNA (Fig. 7.16). MutHand MutL (with ATP bound) then join the complex, which then moves along the DNA in either direction until it finds a hemimethylated GATC motif, which can be as far a few thousand base pairs away. Until this point, the nuclease function of MutH has been dormant, but it is activated in the presence of ATP at a hemimethylated GATC. It cleaves the unmethylated DNA strand, leaving a nick 5' to the G on the strand containing the unmethylated GATC (i.e. the new DNA strand). The same strand is nicked on the other side of the mismatch. Enzymes involved in other processes of repair and replication catalyze the remaining steps. The segment of single-stranded DNA containing the incorrect nucleotide is to be excised by UvrD, also known as helicase II and MutU. SSB and exonuclease I are also involved in the excision. As the excision process forms the gap, it is filled in by the concerted action of DNA polymerase III (Fig. 7.16.).
Figure 7.16 (part 1). Mismatch Repair by MutHLS: recognition of mismatch (shown in red), identifying the new DNA strand (using the hemimethylated GATC shown in blue) and cutting to encompass the unmethylated GATC and the misincorporated nucleotide (red G).
Figure 7.16 (part 2). Mismatch Repair: excision of the DNA with the misincorporated nucleotide bu Uvr D (aided by exonuclease I and SSB), gap filling by DNA polymerase III and ligation.
Mismatch repair is highly conserved, and investigation of this process in mice and humans is providing new clues about mutations that cause cancer.Homologs to the E. coli genes mutLand mutShave been identified in many other species, including mammals. The key breakthrough came from analysis of mutations that cause one of the most common hereditary cancers, hereditary nonpolyposis colon cancer(HNPCC). Some of the genes that, when mutated, cause this disease encode proteins whose amino acid sequences are significantly similar to those of two of the E. colimismatch repair enzymes. The human genes are called hMLH1(for human mutLhomolog 1), hMSH1, and hMSH2(for human mutS homolog 1 and 2, respectively). Subsequent work has shown that these enzymes in humans are involved in mismatch repair. Presumably the increased frequency of mutation in cells deficient in mismatch repair leads to the accumulation of mutations in proto-oncogenes, resulting in dysregulation of the cell cycle and loss of normal control over the rate of cell division.
The human homologs to bacterial enzymes involved in mismatch repair are also implicated in homologous functions. Given the human homologs discussed above, which enzymatic functions found in bacterial mismatch repair are also found in humans? What functions are missing, and hence are likely carried out by an enzyme not homologous to those used in bacterial mismatch repair?
Recombination Repair (Retrieval system)
In the three types of excision repair, the damaged or misincorporated nucleotides are cut out of DNA, and the remaining strand of DNA is used for synthesis of the correct DNA sequence. However, this complementary strand is not always available. Sometimes DNA polymerase has to synthesize past a lesion, such as a pyrimidine dimer or an AP site. One way it can do this is to stop on one side of the lesion and then resume synthesis about 1000 nucleotides further down. This leaves a gap in the strand opposite the lesion (Fig. 7.17).
The information needed at the gap is retrieved from the normal daughter molecule by bringing in a single strand of DNA, using RecA-mediated recombination (see Chapter VIII). This fills the gap opposite the dimer, and the dimer can now be replaced by excision repair (Fig. 7.17). The resulting gap in the (previously) normal daughter can be filled in by DNA polymerase, using the good template.
Figure 7.17. Recombination repair, a system for retrieval of information
As just described, DNA polymerase can skip past a lesion on the template strand, leaving behind a gap. It has another option when such a lesion is encountered, which is to synthesis DNA in a non-template directed manner. This is called translesion synthesis, bypass synthesis, or error-prone repair. This is the last resort for DNA repair, e.g. when repair has not occurred prior to replication. In translesion replication, the DNA polymerase shifts from template directed synthesis to catalyzing the incorporation of random nucleotides. These random nucleotides are usually mutations (i.e. in three out of four times), hence this process is also designated error-prone repair.
Translesion synthesis uses the products of the umuCand umuDgenes. These genes are named for the UV nonmutable phenotype of mutants defective in these genes
Question 7.11. Why do mutations in genes required for translesion synthesis (error prone repair) lead to a nonmutable phenotype?
UmuD forms a homodimer that also complexes with UmuC. When the concentration of single-stranded DNA and RecA are increased (by DNA damage, see next section), RecA stimulates an autoprotease activity in UmuD2 to form UmuD’2. This cleaved form is now active in translesional synthesis. UmuC itself is a DNA polymerase. A multisubunit complex containing UmuC, the activated UmuD’2 and the a subunit of DNA polymerase III catalyze translesional synthesis. Homologs of the UmuC polymerase are found in yeast (RAD30) and humans (XP-V).
A coordinated battery of responses to DNA damage in E. coliis referred to as the SOS response. This name is derived from the maritime distress call, “SOS” for "Save Our Ship". Accumulating damage to DNA, e.g. from high doses of radiation that break the DNA backbone, will generate single-stranded regions in DNA. The increasing amounts of single-stranded DNA induce SOS functions, which stimulate both the recombination repair and the translesional synthesis just discussed.
Key proteins in the SOS response are RecA and LexA. RecA binds to single stranded regions in DNA, which activates new functions in the protein. One of these is a capacity to further activate a latent proteolytic activity found in several proteins, including the LexA repressor, the UmuDprotein and the repressor encoded by bacteriophage lambda (Fig. 7.18). RecA activated by binding to single-stranded DNA is not itself a protease, but rather it serves as a co-protease, activating the latent proteolytic function in LexA, UmuD and some other proteins.
In the absence of appreciable DNA damage, the LexA protein represses many operons, including several genes needed for DNA repair: recA, lexA, uvrA, uvrB, and umuC.When the activated RecA stimulates its proteolytic activity, it cleaves itself (and other proteins), leading to coordinate induction of the SOS regulated operons (Fig. 7.18).
Figure 7.18. RecA and LexA control the SOS response.
The DNA repair systems discussed above operate by surveillance of the genome for damage or misincorporation and then bring in enzymatic machines to repair the defects. Other systems of surveillance in bacterial genomes are restriction/modification systems. These look for foreign DNA that has invaded the cell, and then destroy it. In effect, this is another means of protecting the genome from the damage that could result from the integration of foreign DNA.
These systems for safeguarding the bacterial cell from invasion by foreign DNA use a combination of covalent modification and restriction by an endonuclease. Each species of bacteria modifies its DNA by methylation at specific sites (Fig. 7.19). This protects the DNA from cleavage by the corresponding restriction endonuclease. However, any foreign DNA (e.g. from an infecting bacteriophage or from a different species of bacteria) will not be methylated at that site, and the restriction endonuclease will cleave there. The result is that invading DNA will be cut up and inactivated, while not damaging the host DNA.
Figure 7.19. Restriction/modification systems in bacteria.
Any DNA that escapes the restriction endonuclease will be a substrate for the methylase. Once methylated, the bacterium now treats it like its own DNA, i.e. does not cleave it. This process can be controlled genetically and biochemically to aid in recombinant DNA work. Generally, the restriction endonuclease is encoded at the rlocus and the methyl transferase is encoded at the m locus. Thus passing a plasmid DNA through an r‑m+strain (defective in restriction but competent for modification) will make it resistant to restriction by strains with a wildtype r+gene. For some restriction/modification systems, both the endonuclease and the methyl transferase are available commercially. In these cases, one can modify the foreign DNA (e.g. from humans) prior to ligating into cloning vectors to protect it from cleavage by the restriction endonucleases it may encounter after transformation into bacteria.
For the type II restriction/modification systems, the methylation and restriction occurs at the same, pseudopalindromic site. These are the most common systems, with a different sequence specificity for each bacterial species. This has provided the large variety of restriction endonucleases that are so commonly used in molecular biology.