DNA Repair by Reversal of Damage

Asli Memisoglu, Harvard School of Public Health, Boston, Massachusetts, USA Leona D Samson, Harvard School of Public Health, Boston, Massachusetts, USA
Some DNA repair mechanisms involve the simple reversal of DNA damage. These processes require a single protein that is able first to recognize DNA damage, and then to catalyse the reversal of damage to the DNA, thus restoring the DNA to its original state.

Secondary article
Article Contents
. Introduction . Photoreactivation of DNA . DNA Photolyases in Prokaryotes and Lower Eukaryotes . DNA Photolyases in Higher Organisms . Repair of Spore Photoproducts . Repair of O6-Alkylguanine and O4-Alkylthymine in DNA . The Adaptive Response to Alkylation Damage in E. coli . Repair of Single-Strand Breaks by DNA Ligases . Summary

Introduction
The genetic integrity of all organisms is constantly challenged by exposure to DNA-damaging agents from sources in the environment as well as from those produced endogenously. As a consequence of such persistent threats to the genome, every organism employs several dierent mechanisms to repair DNA damage. Some of the DNA repair pathways are complex, requiring the concerted action of many proteins to recognize and remove segments of damaged DNA, and to resynthesize new DNA to replace that which was removed. In contrast to these rather intricate DNA repair pathways, there are some mechanisms of DNA repair that involve the simple chemical reversal of DNA damage. DNA repair by direct reversal requires a single protein, able to recognize and catalyse the chemical reversal of damage, thus restoring DNA to its original state. Proteins that directly reverse DNA damage include photolyases, O6-methylguanine DNA repair methyltransferases (MTases), the Bacillus subtilis spore photoproduct lyase and DNA ligases. These DNA repair proteins are the subject of this article.

Photoreactivation of DNA
Ultraviolet (UV) irradiation has been a signicant source of DNA damage throughout evolution, inducing covalent bond formation between two adjacent pyrimidine (cytosine or thymine) residues in DNA. The types of covalently linked pyrimidines dimers that are formed by UV exposure include cyclobutane dipyrimidines (CPDs), 6-4 photoproducts and 5-thyminyl-5,6-dihydrothymine dimers (also referred to as spore photoproducts, SP; Figure 1). UV damage to DNA can interfere with the progression and accuracy of DNA replication and thus can result in cell death or mutation, respectively. Although all three types of UV-induced dimers are subject to direct reversal, only CPDs and 6-4 photoproducts are repaired by a visiblelight-dependent, energy-independent process called photoreactivation (PR). PR was discovered in 1949 when it was observed that exposure of the bacterium Streptomyces

griseus to visible light unexpectedly inuenced UV sensitivity. At about the same time, a similar phenomenon was observed in a bacteriophage experimental model system: UV-irradiated bacteriophage were able to reproduce in bacteria that were exposed to visible light much better than in bacteria shielded from such light. Thus, exposure to visible light reactivated the ability of the bacteriophage to productively infect bacteria and this was also referred to as PR. Since its original discovery, the process of PR has been extensively studied and is now known to be due to direct reversal of UV-induced DNA damage by PR enzymes called photolyases. Photolyases use energy from visible light to turn certain pyrimidine dimers into two monomers. PR of CPDs has been the most extensively studied and the molecular mechanism of this reaction has been elucidated in considerable detail. In contrast, less is known about PR of 6-4 photoproducts. CPD (and perhaps all) photolyases are able to capture energy from visible light via two lightabsorbing cofactors. CPD photolyases have avin adenine dinucleotide (FAD) as one cofactor and either 5,10methenyl tetrahydrofolate (MTHF) or 8-hydroxy-5-deazaavin (8-HDF) as the second. One chromophore (either MTHF or 8-HDF) captures a photon of light and then transfers the excitation energy to the other chromophore (FAD). The transfer of energy to FAD in turn initiates a series of electron transfers that ultimately results in monomerization of the CPD (reviewed in Sancar, 1994). Although it has long been known that CPDs are substrates for PR, a photolyase that can act upon the 6-4 photoproduct was more recently discovered in the fruity Drosophila melanogaster. However, for the rest of this article, unless otherwise indicated, the term photolyase refers to the CPD photolyase.

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DNA Repair by Reversal of Damage

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Figure 1 Chemical structure of specific types of DNA base damage subject to direct reversal. (a) Cyclobutane pyrimidine dimer (CPD) produced by UV light. (b) Pyrimidine (6-4) pyrimidone photoproduct ((6-4) photoproduct) produced by UV light. (c) 5-Thyminyl-5,6-dihydrothymine (spore photoproduct) produced specifically in A DNA by UV light. (d) O6MeG produced by methylating agents. (e) O4MeT produced by methylating agents. (f) Potential base pairing of O6MeG with thymine (top) and O4MeT with guanine (bottom).

DNA Photolyases in Prokaryotes and Lower Eukaryotes

PR of UV-induced CPDs has long been known to operate in both Escherichia coli and in the budding yeast Saccharomyces cerevisiae. The genes encoding photolyase have been cloned (the E. coli phr gene and the S. cerevisiae PHR1) and mutants in these genes that render photolyase unable to utilize light energy to stimulate DNA repair are sensitive to the cytotoxic eects and mutagenic eects of UV light. The amino acid sequence of the E. coli and S. cerevisiae photolyases share 34% identity and the two enzymes are functionally interchangeable to a certain extent. More specically, expression of E. coli phr in photolyase-decient phr1 2 S. cerevisiae reverses its UVsensitive phenotype in the presence of visible light, and vice versa. The ability of similar DNA repair proteins from evolutionarily distant organisms to function in heterologous environments indicates that these proteins are very

well conserved throughout evolution, emphasizing their importance. Photolyase-decient E. coli and S. cerevisiae are more sensitive than wild-type to UV-induced cytotoxicity even in the absence of photoreactivating light. Although photolyases have an absolute requirement for visible light in order to catalyse the monomerization of pyrimidine dimers, it turns out that binding of photolyase enzymes to damaged DNA occurs in the absence of visible light and such binding can contribute to UV resistance via a PRindependent process (Sancar and Smith, 1989). This process has been called dark repair. The role of photolyases in dark repair is dependent on a functional DNA nucleotide excision repair pathway (NER) that can repair a wide range of DNA damages. NER involves the excision of a short segment of single-stranded DNA spanning the damaged nucleotides. The resulting gap is lled in by DNA synthesis to replace the excised region using the opposite undamaged strand as template. Although NER exists in all organisms studied thus far, the molecular details vary

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DNA Repair by Reversal of Damage

considerably between prokaryotes and eukaryotes. In the absence of visible light, E. coli photolyase binding appears to facilitate CPD repair via the E. coli NER machinery and such facilitation presumably requires specic protein protein interactions. Although expression of the S. cerevisiae photolyase gene in E. coli confers UV resistance in the presence of visible light, in the absence of light it actually confers UV sensitivity. This sensitization is also dependent on a functional NER pathway and probably reects the fact that while the yeast photolyase can bind CPDs it may not interact productively with the E. coli NER proteins (Sancar and Smith, 1989). Thus, expression of S. cerevisiae PHR1 in phr 2 E. coli inhibits, rather than facilitates, the excision of CPDs in the absence of visible light. Since E. coli and S. cerevisiae photolyases are not interchangeable with regard to dark repair, it seems the protein regions necessary to interact with NER have diverged between the two microbial photolyases. Although photolyase can only monomerize UV dimers, both E. coli and S. cerevisiae photolyase can bind to DNA lesions produced by the cancer chemotherapeutic cisplatin. The contribution of photolyase binding to cisplatin resistance diers between the two microbes. For E. coli binding of its endogenous photolyase to cisplatin DNA lesions provides resistance to cisplatin toxicity. However, for S. cerevisiae, binding of its photolyase to such lesions actually sensitizes cells to cisplatin toxicity. Presumably the E. coli photolyase enhances repair of cisplatin-damaged DNA via NER, but why this does not occur in S. cerevisiae remains unclear. The in vivo role of photolyases may therefore not be limited to repair of UV-damaged DNA, and the biological consequences of photolyase recognition of DNA lesions may not always be benecial.

DNA Photolyases in Higher Organisms

As mentioned, the amino acid sequences of the E. coli and S. cerevisiae photolyase enzymes are quite similar to one another. Based on similarity to the amino acid sequence of the E. coli and S. cerevisiae photolyases, genes encoding predicted photolyases were identied from the bacterium, Streptomyces griseus; the cyanobacterium, Anacystis nidulans; and the plant, Sinapis alba. The hypothesis that these three genes encode photolyases was supported by their ability to reverse the UV sensitivity of photolyasedecient E. coli. In an alternative cloning approach, genes whose expression reversed the UV sensitivity of photolyasedecient E. coli were identied from Carassius auratus (goldsh), D. melanogaster (fruity) and Oryzias latipes (killish) (Yasui et al., 1994). Although the enzymes identied could substitute for E. coli photolyase, sequence analysis indicated that the amino acid sequence of all three enzymes resembled each other but had no similarity to

previously identied photolyases. The amino acid sequence similarity of the goldsh, fruity and killish photolyases was used in turn to clone similar genes from Patters tridactylis (rat kangaroo), Monodelphis domestica (South American opossum) and Methanobacterium thermoautotrophicum (an archaebacterium) (Yasui et al., 1994). Moreover, expression of the rat kangaroo and opossum genes, like that of the goldsh, fruity and killish, was able to reverse the UV sensitivity of photolyase-decient E. coli, suggesting that they encode photolyases. Thus, this group of enzymes dene another class of photolyases in addition to that dened by the original E. coli and S. cerevisiae photolyases. Although the two classes have dierent amino acid sequence, it is possible that the overall protein structure is similar among members of the dierent classes. The above example illustrates one of the advantages of using function instead of amino acid sequence as a criterion to identify genes encoding DNA repair enzymes. It is also important to note that the genes cloned by functional complementation are not limited to genes that encode enzymes having the same biochemical activity as that which is lacking in the host cell. As an example, E. coli that were overexpressing CPD photolyase were used as a host to identify the D. melanogaster gene encoding a 6-4 photolyase (Todo et al., 1996). In addition to CPDs, UV light produces 6-4 photoproducts that are also known to be biologically relevant. A gene whose product gives resistance to the potential toxicity of the 6-4 photoproduct was cloned by using E. coli that were overexpressing CPD photolyase, so that much of the remaining UV-induced cytotoxicity observed would be due to the 6-4 photoproduct rather than to CPDs. Using this strain as a host, a gene whose expression provided UV resistance was identied. This gene was found to encode a unique photolyase that repairs 6-4 lesions. Although the D. melanogaster 6-4 photolyase bears only limited sequence similarity to both E. coli and S. cerevisiae CPD photolyase, it turned out to be quite similar to the plant blue-light photoreceptor family. Homologous genes whose products act as blue-light receptors have been identied from the plants Arabidopsis thaliana and Sinabis alba, but puried proteins do not possess either CPD or 6-4 photolyase activity and expression of each of these genes does not reverse the UV sensitivity of photolyase-decient phr 2 E. coli or yeast. Plant blue-light photoreceptors, also known as cryptochrome photoreceptors, are known to mediate plant growth, owering time and phototropism (growth toward light) in response to blue light. Based on sequence similarity to the D. melanogaster 6-4 photolyase and blue-light photoreceptors, two homologous human genes (CRY1 and CRY2) that are also homologous to one another were identied. Both the CRY1 and CRY2 proteins were found to have spectrophotomeric patterns similar to photolyases and photoreceptors. These patterns
3

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DNA Repair by Reversal of Damage

are due to the light-reactive nature of the chromophores. Thus, CRY1 and CRY2 possess the chromophores necessary for the capture of visible light energy. Since neither CRY1 nor CRY2 possess CPD or 6-4 photoproduct photolyase it was hypothesized that these proteins must function in some other light-sensitive response. It has recently been shown that Cry2 null mice have an altered circadian rhythm as measured by molecular and behavioural markers. Thus CRY2 (and possibly CRY1) somehow contributes to the mammalian circadian rhythm (Thresher et al., 1998).

guanine (O6MeG) or from O4-methylthymine (O4MeT) by all DNA repair MTases, and some bacterial MTases can also transfer methyl groups from the S diasteriomer of methylphosphotriesters (MePT) on the sugar-phosphate backbone to a cysteine residue at a dierent active site (Figure 2). These methyl transfer reactions are irreversible and result in MTase inactivation; thus, the DNA repair MTases have been termed suicide enzymes. DNA repair MTases were rst discovered in E. coli. The genetic and biochemical analysis of O-alkyl repair in E. coli, and characterization of mutant strains altered in MTase activity, demonstrated that O6MeG and O4MeT

Repair of Spore Photoproducts

In addition to CPDs and 6-4 photoproducts, the spore photoproduct (SP) represents a third type of UV-induced pyrimidine dimer in DNA that can also be repaired by direct reversal. SPs are formed in the DNA of metabolically inactive B. subtilis spores and also in dehydrated DNA exposed to UV light. The specicity of SP formation is thought to be due to the uncommon structure of DNA in these two situations aecting the spectrum of DNA lesions produced by UV. Spore DNA (which is partially dehydrated) and in vitro dehydrated DNA assume the A conformation of the DNA double helix, whereas in most physiological conditions DNA is in the B conformation. Upon germination, the majority of SPs are broken down into monomers by an energy-dependent, light-independent reaction that is carried out by the product of the spl gene. Although this reaction is specic for SP and is light independent, the amino acid sequence of the spl gene product bears similarity to DNA photolyases from bacteria and yeast and thus may have some features in common with photolyases. The enzymatic mechanism for SP reversal remains uncertain.

Active MTase

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Repair of O6-Alkylguanine and O4Alkylthymine in DNA

DNA repair methyltransferases in E. coli
Simple alkylating agents can transfer alkyl groups to over a dozen dierent positions on double-stranded DNA. Some alkylated bases cause improper DNA base-pairing and are thus mutagenic. Other alkylated bases can actually prevent cell division by blocking replication by DNA polymerase, which ultimately causes cell death. Methyltransferases (MTases), as their name suggests, transfer methyl groups (as well as larger alkyl groups) from certain oxygens in DNA to an active site cysteine residue, to form Smethylcysteine in the MTase protein itself. More specically, methyl groups can be transferred from O6-methyl4

C5

C5

O O P O
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Phosphodiester

Figure 2 The transfer of methyl groups from DNA by DNA repair MTases. O6MeG, O4MeT and MePT (S-diastereomer only) lesions in doublestranded DNA are recognized by DNA repair MTases and the inappropriate methyl group is transferred to a cysteine residue in the active site of the MTase protein. Methyl transfer inactivates the MTase and so these DNA repair proteins have been called suicide enzymes.

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DNA Repair by Reversal of Damage

DNA lesions cause transition mutations because they have the potential to mispair with thymine and guanine (respectively) during DNA replication. The methyl groups from these bases can be transferred to an active site cysteine in either of two E. coli MTases, namely Ada and Ogt. Ada, but not Ogt, has a second active site cysteine for the transfer of methyl groups from MePT lesions, and methylation at this active site converts the Ada protein into a potent transcriptional activator that is responsible for the adaptive response to alkylating agents in E. coli (Figure 3). E. coli (ada 2 ) are extremely sensitive to the mutagenic and killing eects induced by alkylating agents. Since Ada has a dual role in DNA repair and in regulating genes whose products protect E. coli against alkylation-induced cytotoxicity, it was at rst unclear whether O6MeG/ O4MeT lesions could be cytotoxic as well as mutagenic. However, since Ogt only repairs O6MeG and O4MeT, and is not known to regulate any genes, characterization of ogt mutants revealed that O6MeG and/or O4MeT can indeed cause E. coli cell death. It is now known that O6MeG/ O4MeT-mediated cell death involves the postreplicative mismatch repair pathway in a somewhat unexpected way. It turns out that under certain circumstances the mismatch repair pathway can erroneously process O6MeG lesions, and such processing is thought to create lethal DNA strand

breaks. Thus, mismatch repair-decient cells are actually more resistant to O6MeG/O4MeT-mediated cell death and are described as methylation tolerant. As mentioned, the Ada protein has two active site cysteine residues. Cys-69 is located in the amino-terminal portion of the protein and can transfer methyl groups from MePT. Cys-321 is located in the carboxy-terminal portion of Ada and can transfer methyl groups from either O6MeG or O4MeT. The N-terminal and C-terminal domains are distinct and can function independently. These two domains are connected by a hinge-like structure that is sensitive to cleavage by proteolytic enzymes. Although the tertiary structure of the entire Ada protein has not yet been solved, the structure of each separate domain has been elucidated. The amino-terminal portion, containing Cys69, has been shown to have a tightly associated zinc atom that is important for proper protein folding of Ada and is also thought to be important for the switch between the role of Ada as a DNA repair protein to its role as a transcriptional activator. Resolution of the structure of the C-terminal portion of Ada revealed that Cys-321 is embedded within the molecule; it was therefore hypothesized that a conformational change must take place in order for alkyl transfer to occur. Indeed, a conformational change upon DNA interaction has been observed in the human MTase enzyme.

Uninduced aidB ada alkB alkA

Me

Methyl transfer from damaged DNA to Ada protein

Me Sensing alkylated DNA damage

Transcriptional activator

Transcriptional activation Me Me Me

aidB

ada

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alkA Alkylation resistance

Figure 3 The adaptive response to methylating agents. Methyl transfer from a MePT to cysteine-69 on E. coli Ada turns Ada into a potent transcriptional activator that increases expression from the ada gene as well as from the alkA gene, which encodes the DNA repair enzyme, 3-methyladenine DNA glycosylase, and two other genes, alkB and aidB, which are also thought to play a role in cellular resistance to alkylating agents.

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DNA Repair by Reversal of Damage

DNA repair methyltransferase in mammalian cells

Although O6MeG and O4MeT were shown to be both mutagenic and cytotoxic in E. coli, the biological consequences of these two DNA lesions in mammalian cells was unclear. The ability of the lesions to cause mutation in E. coli depends on how they are perceived by the E. coli DNA replication machinery. If the DNA replication machinery perceives an O6MeG as an adenine homologue, thymine would be paired opposite O6MeG, resulting in transition mutations. It therefore follows that the mutagenicity of O6MeG and O4MeT in other organisms must depend in part upon how the DNA replication complex in those organisms reacts to this kind of DNA template damage. Before the human or mouse MTase had been cloned, isogenic mammalian cell lines diering only in their expression of DNA repair MTase were generated by introducing the E. coli ada gene into cultured human and rodent cells lacking endogenous MTase activity. Acquisition of Ada MTase activity conferred tremendous resistance to the mutagenic, the cytotoxic and the chromosomedamaging eects of a variety of dierent alkylating agents, indicating that the O-alkyl lesions repaired by Ada do indeed produce biological consequences in mammalian cells. Since Ada has two active sites, one for O6MeG/ O4MeT repair and a second for MePT repair, it was important to determine the relative contribution of each active site to providing alkylation resistance. Expression of an active Ada protein fragment, containing just MePT repair activity, demonstrated that MePTs are not toxic to mammalian cells. In contrast, the repair of O6MeG/ O4MeT by the carboxy portion conferred full resistance to alkylation-induced mutation, cell death and chromosome damage. Additional support for this conclusion came from the fact that expression of the E. coli Ogt MTase (which does not repair MePTs) in murine cells conferred resistance to alkylation-induced killing and mutation. Moreover, when expression of the cloned human MGMT MTase cDNA was later achieved in MTase-decient mammalian cells, it was found to confer the same alkylation-resistant phenotype as Ada and Ogt; since the mammalian MTases do not repair MePT and since they are extremely inecient at repairing O4MeTs, it seems likely that O6MeG is primarily responsible for mutation, cell death and chromosome damage in alkylated mammalian cells. Recent data indicate that unrepaired O6MeG lesions can trigger apoptosis (also called programmed cell death) and this characteristic may account for some of the cytotoxicity caused by O6MeG. As in E. coli, the cytotoxicity of O6MeG in mammalian cells depends on a functional mismatch repair pathway (Karran and Marinus, 1982).

DNA repair methyltransferase in whole animals

Studies on carcinogenesis in whole animals provide evidence that DNA repair MTase activity is an important factor in the prevention of alkylation-induced cancer. Bacterial and human MTases have been expressed in transgenic mice. Expression of E. coli Ada, resulting in an 8-fold increase in hepatic MTase activity, reduced the number of liver tumours induced by nitrosamines, a class of alkylating agent. Similarly, targeted expression of human MGMT to the thymus and colon prevented alkylation-induced thymic lymphomas and reduced the induction of putative preneoplastic colon lesions. In the transgenic mouse studies described above, MTase was overexpressed in certain organs. The generation of DNA repair MTase null mice has provided a tool to determine the importance of DNA repair MTase for cancer induction of all tissues. Targeted disruption of the Mgmt gene in mice has conrmed its importance in providing resistance against the killing and tumorigenic eects of alkylating agents. Mgmt null mice have the following phenotypes: growth retardation; sensitivity to the killing eects of the methylating agent methylnitrosourea (MNU) and several agents used for cancer chemotherapy, including 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), streptozotocin and temozolomide; and sensitivity to tumour induction by MNU treatment (Tsuzuki et al., 1996; Glassner et al., 1999). Severe damage to a number of organs was observed in Mgmt 2 / 2 mice treated with MNU, with the most dramatic eects observed in organs relating to haematopoiesis. There were fewer lymphocytes in the thymus and fewer haematopoietic stem cells (that give rise to several dierentiated cells of the immune system) within the bone marrow in Mgmt 2 / 2 mice treated with both MNU and various chemotherapeutic alkylating agents. Mgmt 2 / 2 mice were also more sensitive than wild-type to the induction of tumours by MNU, specically thymic lymphomas and lung adenomas. Other tissues that became particularly vulnerable to DNAdamaging agents upon DNA repair MTase removal were the intestines. MNU-treated Mgmt 2 / 2 mice had dyplastic (abnormal in size, shape and/or organization) cells within the small and large intestines. Malnourishment, owing to the inability of the damaged intestines to absorb nutrients, may explain the growth retardation that is observed in Mgmt 2 / 2 mice and may contribute to their alkylation sensitivity. These data underscore the important role that DNA repair plays in preventing a normal cell from being transformed into a cancer cell after exposure to exogenous DNA-damaging agents.

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DNA Repair by Reversal of Damage

DNA repair methyltransferase in cancer treatment

The observation that DNA repair MTase activity is a strong determinant of cell sensitivity to certain agents used in cancer chemotherapy (e.g. BCNU, temozolamide, streptozotocin) has led to the development of two strategies for modulating DNA repair MTase activity in an eort to enhance the eectiveness, and reduce the risks, of chemotherapeutic regimens. During cancer chemotherapy, the objective is to administer the drug, such that all the cancer cells are killed whereas enough of the noncancer cells survive such that the short- and long-term side eects are tolerable. In one approach, attempts are being made to sensitize cancer cells to alkylating agent chemotherapy by inhibiting DNA repair MTase activity with O6-benzylguanine (O6BeG), a potent inhibitor of mammalian DNA repair MTase. O6BeG acts as a substrate for alkyl transfer by the DNA repair MTase. The DNA repair MTase is irreversibly inactivated upon transfer of the benzyl group to the acceptor cysteine residue within the protein. Several derivatives of O6BeG have been developed and combined treatment of isolated tumour cells with O6BeG and its derivatives dramatically increased the sensitivity of these cells to killing by BCNU. Certain tumours, such as gliomas, are very resistant to treatment by the nitrosourea class of alkylating agents and clinical trials are currently underway to determine if administration of O6BeG to patients with malignant glioma enhances the responsiveness of the cancer cells to BCNU chemotherapy (reviewed in Dolan and Pegg, 1997). The second clinical approach is to increase MTase activity and thus alkylation resistance in the dose-limiting tissues. Such increases should raise the maximum tolerated dose of the chemotherapeutic and might be expected to decrease long-term side eects, namely therapy-related leukaemias. The dose of BCNU that can be administered for the treatment of malignant brain tumours is limited by the most sensitive tissue, namely the bone marrow. In short, BCNU treatment can cause death, due to bone marrow ablation at doses that do not eectively ablate the tumour. Recombinant tools have been generated for gene therapy aimed at expressing extra DNA repair MTase in bone marrow progenitor cells. Such gene therapy has already been tested in mice and it was observed that mice transplanted with bone marrow cells expressing the MTase gene could survive larger doses of BCNU than control mice (Maze et al., 1996). This approach could be used to increase the therapeutic window for BCNU treatment of certain cancers, and human MTase gene therapy is currently in Phase I clinical trials.

The Adaptive Response to Alkylation Damage in E. coli

In 1977, it was discovered that E. coli exposed to sublethal doses of the alkylating agent, N-methyl-N-nitro-N-nitrosoguanidine (MNNG), became extremely resistant to the lethal and mutagenic eects of subsequent exposure to high doses of MNNG (Samson and Cairns, 1977). This phenomenon was coined the adaptive response to alkylating agents and involves the Ada DNA repair MTase. Upon transfer of a methyl group from the S diasteriomer of MePT, Ada becomes a powerful transcriptional activator and increases expression of four genes, namely ada, alkA, alkB, aidB (Figure 3). AlkA encodes a 3-methyladenine DNA glycosylase that is known to initiate base excision repair of potentially lethal 3-methyladenine residues, whereas the precise involvement of the AlkB and AidB proteins in methylation resistance is not yet understood. The induced transcription of ada, alkA, alkB and aidB by the Ada protein confers tremendous alkylation resistance upon E. coli. Thus the repair of alkylated DNA by the Ada protein acts both as a sensor of DNA alkylation damage and as an activator of the expression of its own gene, that of another known DNA repair enzyme and two other genes whose products are likely to play a role in DNA repair. Ada also seems to be involved in turning o the adaptive response. Unmethylated Ada was found to inhibit ada gene induction by methylated Ada. Thus once the repair of alkylated DNA is complete, the newly made, unmethylated Ada proteins remain unmethylated and provide a signal for turning o the adaptive response. Although transcription of both the ada and alkA genes is regulated by the Ada protein, there are distinct dierences in the regulation of each gene. Certain truncated forms of the Ada protein support methylation-induced expression of the alkA gene similar to the wild-type protein; the same truncated Ada proteins support constitutive Ada expression that is unresponsive to methylation induction. Furthermore, although unmethylated Ada protein inhibits transcriptional induction of the ada gene, it had no eect on the transcriptional induction of the alkA gene by methylated Ada. Although there have been some reports of an adaptive response to various toxicants in yeast and mammalian cells, there does not appear to be a MTase-regulated pathway similar to that observed in E. coli.

Repair of Single-Strand Breaks by DNA Ligases

In addition to their role as the nal enzymatic step of several DNA excision repair pathways, DNA ligases are also thought to directly repair single-strand breaks in DNA

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DNA Repair by Reversal of Damage

in much the same way that adjacent Okazaki fragments are sealed together. Single-strand breaks in DNA can be caused by ionizing radiation, alkylating agents and oxidizing agents. Only single-strand DNA breaks where one terminus has a 3-hydroxyl and the other has a 5phosphate are subject to DNA ligation. Such hydrolytic strand breaks are known to represent a fraction of the total single-strand breaks produced by ionizing irradiation. Indeed, treatment of irradiated DNA containing singlestrand breaks with E. coli DNA ligase I was observed to reduce the number of free 5-phosphate ends, indicating that these ends were joined by DNA ligase treatment. However, it is unclear whether DNA ligase directly repairs such DNA single-strand breaks in vivo.

Summary
Mechanisms of DNA repair by direct reversal are important for the survival of an organism and for the maintenance if its genomic integrity. Since such mechanisms involve a single step carried out by a single protein (rather than multiple steps carried out by numerous proteins and protein complexes), DNA repair by direct reversal is extremely quick and ecient. Studies on the genes encoding proteins involved in direct reversal of DNA damage, plus the biochemical characterization of such proteins, have led to a deeper understanding of mammalian carcinogenesis and to the development of tools to enhance the eectiveness of cancer chemotherapeutic regimens. Finally, studies of DNA repair by direct reversal in microbes, sh, plants and insects have unexpectedly led to the discovery of a mammalian gene whose product is involved in the mammalian circadian rhythm.

Glassner BJ, Weeda G, Allan J et al. (1999) DNA repair methyltransferase (Mgmt) knockout mice are sensitive to the lethal eects of chemotherapeutic alkylating agents. Mutagenesis 14: 100109. Karran P and Marinus MG (1982) Mismatch correction at O6Methylguanine residues in E. coli DNA. Nature 296(5860): 868869. Maze R, Carney JP, Kelley MR et al. (1996) Increasing DNA repair methyltransferase levels via bone marrow stem cell transduction rescues mice from the toxic eects of 1,3-bis(2-chloroethyl)-1nitrosourea, a chemotherapeutic alkylating agent. Proceedings of the National Academy of Sciences of the USA 93: 206210. Samson L and Cairns J (1977) A new pathway for DNA repair in Escherichia coli. Nature 267(5608): 281283. Sancar A (1994) Structure and function of DNA photolyase. Biochemistry 33(1): 29. Sancar GB and Smith FW (1989) Interactions between yeast photolyase and nucleotide excision repair proteins in Saccharomyces cerevisiae and Escherichia coli. Molecular and Cellular Biology 9(11): 47674776. Thresher RJ, Vitaterna MH, Miyamoto Y et al. (1998) Role of mouse cryptochrome blue-light photoreceptor in circadian photoresponses. Science 282: 14901494. Todo T, Ryo H, Yamamoto K et al. (1996) Similarity among the Drosophila (6-4) photolyase, a human photolyase homolog, and the DNA photolyase-blue-light photoreceptor family. Science 272: 109 112. Tsuzuki T, Sakumi K, Shiraishi A et al. (1996) Targeted disruption of the DNA repair methyltransferase gene renders mice hypersensitive to alkylating agent. Carcinogenesis 17: 12151220. Yasui A, Eker AP, Yasuhira S et al. (1994) A new class of DNA photolyases present in various organisms including aplacental mammals. EMBO Journal 13(24): 61436151.

Further Reading
Friedberg EC, Walker GC and Siede W (1995) DNA repair by reversal of damage. In: DNA Repair and Mutagenesis, pp. 91133. Washington, DC: American Society for Microbiology Press. Lindahl T, Sedgwick B, Sekiguchi M and Nakabeppu Y (1988) Regulation and expression of the adaptive response to alkylating agents. Annual Review of Biochemistry 57: 133157. Pieper RO (1998) Cellular responses to methylation damage. In: Nickolo JA and Hoekstra MF (eds) DNA Damage and Repair: DNA Repair in Higher Eukaryotes, pp. 3349. Totowa, NJ: Humana Press. Yasui A and Eker APM (1998) DNA photolyases. In: Nickolo JA and Hoekstra MF (eds) DNA Damage and Repair: DNA Repair in Higher Eukaryotes, pp. 932. Totowa, NJ: Humana Press.

References
Dolan ME and Pegg AE (1997) O6-Benzylguanine and its role in chemotherapy. Clinical Cancer Research 3: 837847.

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