7.4: Repair Mechanisms (2023)

damage reversal

Some types of covalent base change in DNA can be reversed directly. This occurs through specific enzyme systems that recognize the altered base and break the bonds to remove the adduct or return the base to its normal structure.

photoreactivationis a light-dependent process used by bacteria to reverse pyrimidine dimers formed by UV radiation. The photolyase enzyme 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 the DNA. Note that this is not formally the opposite of the reaction that formed the pyrimidine dimers, since visible light energy is used to break the bonds between the pyrimidines and no UV radiation is released. However, the result is that the DNA structure has returned to its pre-UV damage state. The enzyme photolyase has two subunits, which are encoded by thePhrAyPhrBgenes meE. coli.

A second example of damage reversal is theremoval of methyl groups. For example, the enzymeO6-methylguanine methyltransferase, encoded byadagene andE. coli, to recognizeO6-methylguanine in the DNA duplex. It then removes the methyl group, transferring it to an amino acid in the enzyme. The methylated enzyme is no longer active, so this has been said to be a suicide mechanism of the enzyme.

excision repair

The most common means of repairing damage or a mismatch is to cut it from the duplex DNA and recopy the remaining complementary strand of DNA, as illustrated in Figure 7.12. Three different types of excision repair have been characterized: nucleotide excision repair, base excision repair, and mismatch repair. They all use onecut, copy and pastemechanism. NocutIn this step, an enzyme or complex removes a damaged base or nucleotide chain from the DNA. For thecopying, a DNA polymerase (DNA polymerase I inE. coli) will copy the template to replace the damaged and removed wire. DNA polymerase can initiate synthesis from 3' OH intA single-stranded break (cut) or gap in DNA that remains at the site of damage after excision. Finally, ingluedAt this stage, DNA ligase seals the remaining cut to provide intact and repaired DNA.

Nucleotide Excision Repair (NER)

Emnucleotide excision repair, the damaged bases are excised within a nucleotide strand and replaced with DNA according to the instructions of the undamaged template strand. This repair system is used to remove pyrimidine dimers formed by UV radiation, as well as nucleotides modified by bulk chemical adducts. The common feature of the damage that is repaired by nucleotide excision is that the modified nucleotides cause significant distortion in the DNA helix. NER occurs in nearly all organisms examined.

Some of the best characterized enzymes that catalyze this process are UvrABC excynuclease and UvrD helicase inE. coli.Genes encoding this repair function were found to be mutants that are highly sensitive to UV damage, indicating that the mutants are defective in UV repair. As illustrated in Figure 7.13, wild typeE. colicells are only killed by higher doses of UV radiation. Mutant strains that are substantially more sensitive to UV radiation can be identified; these are defective in functions necessary forultraviolet-rresistance, abbreviatedUVR. By collecting large numbers of these 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, code for proteins that play important roles in NER; they areuvrA, uvrB,uvrCyuvrd.

The enzymes encoded byUVRGenes have been studied in detail. The polypeptide products of theuvra,uvrB, youuvrCGenes are subunits of a multisubunit enzyme calledExcinucleasa UvrABC. UvrA is the protein encoded byuvra, UvrB is encoded byuvrB, etc. The UvrABC complex recognizes structural distortions induced by DNA damage, such as pyrimidine dimers. It then splits on both sides of the damage. So UvrD (also called helicase II), the product ofuvrdgene, unwinds the DNA, releasing the damaged segment. Thus, for this system, the UvrABC and UvrD proteins perform a series of steps in the cutting phase of excision repair. This leaves an empty substrate for copying by DNA polymerase and pasting by DNA ligase.

UvrABC proteins form a dynamic complex that recognizes damage and performs endonucleolytic cleavage on both sides. The two cuts around the damage allow the single-stranded segment containing the damage to be excised by UvrD helicase activity. Therefore, the dynamic UvrABC complex and the UvrBC complex can be calledexcinucleases. After the damaged segment is removed, a gap of 12 to 13 nucleotides remains in the DNA. This can be filled in with DNA polymerase and the remaining notch can be sealed with DNA ligase. Since the undamaged template directs synthesis by DNA polymerase, the resulting duplex DNA is no longer damaged.

In more detail, the process is as follows (Figure 7.14). UVA₂(a dimer) and UvrB recognize the damaged site as a (UvrA)2UvrB complex. UVA₂it then dissociates, in a step that requires ATP hydrolysis. This is an autocatalytic reaction as it is catalyzed by UvrA which is an ATPase. Upon dissociation of UvrA, UvrB (at the damaged site) forms a complex with UvrC. The UvrBC complex is the active nuclease. Make the incisions on both sides of the damage, another step that requires ATP. The phosphodiester backbone is cleaved 8 nucleotides on the 5' side of the damage and 4-5 nucleotides on the 3' side. Finally, UvrD helicase unwinds the DNA to remove the damaged segment. The damaged DNA segment is dissociated bound to the UvrBC complex. Like all helicase reactions, unwinding requires ATP hydrolysis to break the base pairs. Therefore, the hydrolysis of ATP is required in three steps of this series of reactions.

Nucleotide excision repair is highly active in mammalian cells as well as in cells of many other organisms. The DNA of a normal skin cell exposed to sunlight would accumulate thousands of dimers a day if this repair process did not remove them! A human genetic disease called xeroderma pigmentosum (XP) is a skin condition caused by a defect in the enzymes that eliminate UV damage. Fibroblasts isolated from individual XP patients are markedly sensitive to UV radiation when cultured, similar to the phenotype exhibited byMI.coliUVRmutants. These XP cell lines can be fused in culture and tested for their ability to restore resistance to UV damage. XP cell lines that do this fall into different complementation groups. Several complementation groups, or genes, have been defined in this way. Considerable progress has recently been made in identifying the proteins encoded by each XP gene (Table 7.2). Note the close analogy to the bacterial functions required for NER. Similar functions are also found in yeast (Table 7.2). Additional proteins used in eukaryotic NER include hHR23B (which forms complexes with the XPC DNA damage sensor), ERCCI (which forms complexes with XPF to catalyze the 5' incision to the damage site), the various other subunits of TFIIH (see Chapter 10) and the single-chain RPA binding protein.

NER occurs in two modes in many organisms, including bacteria, yeast, and mammals. One is global repair that works across the entire genome, and the second is a specialized activity that is associated with transcription. Most of the XP gene products listed in Table 2 function in both NER modes in mammalian cells. However, XPC (acting in a complex with another protein called hHR23B) is a DNA damage sensor specific to the global NER genome. In transcription-coupled NER, elongating RNA polymerase is arrested at a lesion in the template strand; perhaps this is the damage recognition activity for this NER mode. One of the basal transcription factors associated with RNA polymerase II, TFIIH (see Chapter 10), also plays a role in both types of NER. A rare genetic disease in humans, Cockayne syndrome (CS), is associated with a specific defect in transcription-coupled repair. Two complementation groups were identified,CSAyCSB. Determining the nature and activity of the proteins encoded by them will provide additional information on the efficient repair of transcribed DNA strands. The phenotype of patients with CS is pleiotropic, exhibiting photosensitivity and severe neurological and other developmental disorders, including premature aging. These symptoms are more severe than those seen in XP patients with no detectable NER, indicating that transcription-coupled repair or CS proteins have functions in addition to those of NER.

Other genetic diseases also result from a deficiency in DNA repair function, such as Bloom syndrome and Fanconi anemia. These are intensive areas of current research. A good resource for up-to-date information on these and other inherited disorders, as well as human genes in general, is Online Mendelian Inheritance in Man, or OMIM, accessible athttp://www.ncbi.nlm.nih.gov.

Ataxia telangiectasia, or AT, illustrates the effect of changes in a protein that is not directly involved in repair, but perhaps indicates that it is required for proper DNA repair. AT is a rare, recessive genetic disorder characterized by irregular gait (ataxia), dilated blood vessels (telangiectasia) in the eyes and face, cerebellar degeneration, progressive mental retardation, immune deficiencies, premature aging, and an approximately 100-fold increase in susceptibility to cancer . This latter phenotype is generating much interest at this locus, as heterozygotes, who comprise about 1% of the population, are also at increased risk of cancer and may account for up to 9% of breast cancers in the United States. the gene that ismetroused inEM(hence the name "ATM") was isolated in 1995 and located on chromosome 11q22-23.

The ATM gene does not appear to encode a protein directly involved in DNA repair (unlike the genes that cause XP after mutation). Instead, AT is caused by a defect in a cell signaling pathway. Based on homologies with 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 with DNA-dependent protein kinases, which require breaks, cuts, or gaps to bind to DNA (via the Ku subunit); DNA binding is required for protein kinase activity. This suggests that the ATM protein may be involved in directing the repair machinery to such damage.

Base split repair

Base excision repair differs from nucleotide excision repair in the types of substrates recognized and the initial cleavage event. Unlike NER, the base excision machinery recognizes damaged bases that do not cause significant distortion in 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 can also remove some damaged bases that distort DNA, such as methylated purines. In general, the initial recognition is a specific damaged base, not a helical distortion in the DNA. A second important difference is that the initial cleavage is directed at the glycosidic bond that connects the purine or pyrimidine base to a deoxyribose in the DNA. This contrasts with the initial cleavage of a phosphodiester bond in NER.

Cells contain a large amount ofglucosilasasthat recognize damaged or inappropriate bases, such as uracil, in DNA. Glycosylase removes the damaged or inappropriate base by catalyzing the cleavage of the N-glycosidic bond that joins the base to the sugar-phosphate backbone. For example, uracil-N-glycosylase, the product ofa littlegene, recognizes uracil in DNA and cuts the N-glycosidic bond between base and deoxyribose (Figure 7.15). Other glycosylases recognize and cleave damaged bases. For example, methylpurine glycosylase removes methylated G and A from DNA. The result of the activity of these glycosylases is an apurinic/apyrimidine site, or AP site (Figure 7.15). At an AP site, the DNA remains an intact duplex, that is, there are no breaks in the phosphodiester backbone, but a base is missing.

then aaccess pointendonucleasait cuts the DNA just 5' from the AP site, thus providing a primer for DNA polymerase. InE. coli, the 5' to 3' exonuclease function of DNA polymerase I removes the damaged region and fills it in with the correct DNA (using the 5' to 3' polymerase, driven by the undamaged complementary strand sequence).

Additional mechanisms have been developed to keep the U's out of the DNA.MI.colialso has a dUTPase, encoded byI havegene, which catalyzes the hydrolysis of dUTP to dUMP. The dUMP product is the substrate for thymidylate synthetase, which catalyzes the conversion of dUMP to dTMP. This keeps the concentration of dUTP in the cell low, reducing the possibility of it being used in DNA synthesis. Thus, the combined action of the products ofI have+a littlegenes helps prevent the accumulation of U no DNA.

misalignment repair

The third type of excisional repair that we will consider isincompatibility fix, 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 approximately once in every 108 bp synthesized inE. coli. However, the mutation rate measured in bacteria is as low as one error per 1010 or 1011 bp. The enzymes that catalyzedisagreementto repairare responsible for this last degree of accuracy. They recognize misincorporated nucleotides, remove them, and replace them with the correct nucleotides. Unlike nucleotide excision repair, mismatch repair does not operate on bulky adducts or large distortions in the DNA helix. Most incompatibilities are substitutes within a chemical class, e.g. a C is incorporated instead of a T. This only causes a subtle helical distortion in the DNA, and the misincorporated nucleotide is a normal component of DNA. A cell's ability to recognize a mismatch reflects the exquisite specificity ofCap, which can distinguish normal base pairs from those resulting from incorrect incorporation. Obviously, the repair mechanism needs to know which of the nucleotides in a mismatched pair is the correct one and which was incorporated incorrectly. It does this by determining which string was most recently synthesized and repairing the mismatch in the nascent string.

EmE. coli, methylation of A in a GATC motif provides a covalent marker for the parental strand, so DNA methylation is used to discriminate the parental strands from the progeny. Remember that theJackmetilasecatalyzes the transfer of a methyl group to the A of the GATC pseudopalindromic sequence in duplex DNA. Methylation is delayed several minutes after replication. In this interval before methylation of the new strand of DNA, the mismatch repair system can find mismatches and direct its repair activity to nucleotides in the newly replicated unmethylated strand. Therefore, replication errors are preferentially removed.

The MutH-MutL-MutS, or MutHLS, enzyme complex catalyzes the repair of mismatches inE. coli. The genes that encode these enzymesmuth,felizycap, were discovered because mutation-carrying strains have a high frequency of new mutations. This is calledmutant phenotype, hence the nameperiodwas given to these genes. Not all mutated genes are involved in mismatch repair; for example, mutations in the gene encoding the DNA polymerase III proofreading enzyme also have a mutant phenotype. This gene was independently discovered in DNA replication defect screens (etc) and mutated genes (MutD). Three complementation clusters within the mutant allele pool have been implicated primarily in mismatch repair; these aremuth,felizycap.

Capit will recognize seven of eight possible mismatched base pairs (except C:C) and bind to that site on the DNA duplex (Figure 7.16).MutHyfeliz(with ATP bound) then binds to the complex, which then moves along the DNA in any direction until it encounters a hemimethylated GATC motif, which can be a few thousand base pairs away. Up to this point, the nuclease function of MutH has been inactive, but it is activated in the presence of ATP in a hemimethylated GATC. Cut the unmethylated DNA strand, leaving a notch 5' to the G on the strand containing the unmethylated GATC (ie, the new DNA strand). The same wire is cut across the mismatch. Enzymes involved in other repair and replication processes catalyze the remaining steps. The single-stranded DNA segment containing the incorrect nucleotide must be excised by UvrD, also known as helicase II and MutU. SSB and exonuclease I are also involved in cleavage. As the cleavage process forms the gap, it is filled in by the combined action of DNA polymerase III (Figure 7.16).

Mismatch repair is highly conserved, and investigation of this process in mice and humans is providing new insights into cancer-causing mutations.E. coligenesfelizycapthey have been identified in many other species, including mammals. The main advance came from the analysis of mutations that cause one of the most common hereditary cancers,hereditary non-polyposis 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 theMI.colimismatch repair enzymes. Human genes are calledhMLH1(for humansfelizcounterpart 1),hMSH1, youhMSH2(for humanscaphomologues 1 and 2, respectively). Subsequent work has shown that these enzymes in humans are involved in mismatch repair. Presumably, increased mutation frequency in mismatch repair-deficient cells leads to the accumulation of mutations in proto-oncogenes, resulting in cell cycle dysregulation and loss of normal control over the rate of cell division.

Recombination repair (system recovery)

In all three types of excision repair, damaged or misincorporated nucleotides are removed from the DNA and the remaining strand of DNA is used for synthesis of the correct DNA sequence. However, this complementary aspect is not always available. Sometimes DNA polymerase needs to be synthesized after injury, such as a pyrimidine dimer or an AP site. One way to do this is to stop on one side of the lesion and resume synthesis about 1000 nucleotides down. This leaves a gap in the wire opposite the lesion (Figure 7.17).

The necessary information in the gap is retrieved from the normal daughter molecule by introducing a single strand of DNA by RecA-mediated recombination (see Chapter VIII). This fills in the space opposite the dimer, and the dimer can now be replaced by excision repair (Figure 7.17). The resulting gap in the (formerly) normal daughter can be filled in with DNA polymerase, using the good template.

translation synthesis

As just described, DNA polymerase can bypass a damage to the template strand, leaving a gap. You have another option when encountering such a lesion, which is to synthesize the DNA in a non-template-directed manner. Calledtranslation synthesis, bypass synthesis or error-prone repair. This is a last resort for DNA repair, e.g. when repair did not occur before replication. In translesion replication, DNA polymerase switches from template-directed synthesis to catalyzed random incorporation of nucleotides. These random nucleotides are usually mutations (i.e. three out of four times), so this process is also called error-prone repair.

The translesion synthesis uses the products of theumuCyumuDgenes These genes get their name from thetuV noemtable of phenotypes of mutants defective in these genes

UmuD forms a homodimer that also forms a complex with UmuC. When the concentration of single-stranded DNA and RecA are increased (for DNA damage, see next section), RecA stimulates an autoprotease activity in UmuD.₂para formar UmuD'₂. This cleaved form is now active in translesional synthesis. UmuC itself is a DNA polymerase. A multi-subunit complex containing UmuC, the activated UmuD'₂and the a subunit of DNA polymerase III catalyzes translesional synthesis. UmuC polymerase homologs are found in yeast (RAD30) and humans (XP-V).

the SOS reply

A coordinated battery of DNA damage responses inMI.coliis known as SOS response. This name is derived from the maritime distress call, "SOS" for "Save our ship". Accumulation of DNA damage, e.g. High doses of radiation that break the DNA backbone will generate single-stranded regions in the DNA. Increasing amounts of single-stranded DNA induce SOS functions, which stimulate both the recombinant repair and the translesional synthesis just discussed.

The key proteins in the SOS response areRecAyLexA. RecA binds to single-stranded regions of DNA, activating new functions in the protein. One of these is the ability to further activate a latent proteolytic activity found in several proteins, including the LexA repressor, theUmuDprotein and the repressor encoded by bacteriophage lambda (Figure 7.18). RecA activated by binding to single-stranded DNA is not itself a protease but acts as a coprotease, activating 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 necessary for DNA repair:recA, lexA, uvrA, uvrB from uMC.When activated RecA stimulates its proteolytic activity, it cleaves itself (and other proteins), leading to the coordinated induction of SOS-regulated operons (Figure 7.18).

Restriction/Modification Systems

The DNA repair systems discussed above operate by monitoring the genome for damage or incorrect incorporation and then bringing in enzymatic machinery to repair the defects. Other surveillance systems in bacterial genomes arerestriction/modification systems. They look for foreign DNA that has invaded the cell and then destroy it. In effect, this is another means of protecting the genome from damage that can result from the integration of foreign DNA.

These systems for protecting the bacterial cell from foreign DNA invasion use a combination of covalent modification and restriction by an endonuclease. Each species of bacteria modifies its DNA bymethylationin specific locations (Figure 7.19). This protects the DNA from cleavage by the correspondingrestriction endonuclease. However, any foreign DNA (for example, from an infecting bacteriophage or a different species of bacteria) will not be methylated at that site and the restriction endonuclease will be cleaved there. The result is that the invading DNA will be cut and inactivated without damaging the host DNA.

Any DNA that escapes the restriction endonuclease will be a substrate for the methylase. Once methylated, the bacteria starts to treat it as if it were its own DNA, that is, it does not cleave it. This process can be genetically and biochemically controlled to aid the work of recombinant DNA. In general, restriction endonuclease is encoded in therlocus and methyltransferase is encoded in themetroplace. Thus, by passing a plasmid DNA through ar-m+strain (defective by restriction but competent by modification) will make it resistant to restriction by strains with a wild typer+gene. For some restriction/modification systems, both endonuclease and methyltransferase are commercially available. In such cases, foreign (eg, human) DNA can be modified prior to ligation into cloning vectors to protect it from cleavage by restriction endonucleases that may be encountered after transformation into bacteria.

For type II restriction/modification systems, methylation and restriction occur at the same pseudopalindromic site. These are the most common systems, with a different sequence specificity for each bacterial species. This provided the wide variety of restriction endonucleases that are so frequently used in molecular biology.

Collaborators and Assignments

• Ross C. Hardison, T. Ming Chu Professor deBiochemistry and Molecular Biology(Pennsylvania State University)