The persistence of DNA adducts is the result, for the most part, of the failure of DNA repair, so that its structure returns to normal without evidence of alteration. The structural alterations that may occur in the DNA molecule as a result of interaction with reactive chemical species or di- rectly with ultraviolet or ionizing radiation are considerable. A number of the more frequently seen structural changes in DNA are schematically represented in Figure 3.15. The reaction with chemical species produces adducts on bases, sugars, and the phosphate backbone. Bifunctional reactive chemicals may also cause the crosslinking of DNA strands through reaction with two opposing bases. Other structural changes, such as the pyrimidine dimer formation, are specific for ultraviolet radiation (see below), whereas double-strand DNA breaks are most commonly seen with ionizing radiation (see below). On the other hand, most of the remaining lesions dem- onstrated in Figure 3.15 may occur as a result of either chemical or radiation effects on the DNA molecule. In order to cope with so many structurally different types of DNA damage, a variety of mechanisms have evolved in living cells to deal with each of the types of damage shown in Figure 3.15. A summary of the types of DNA repair most commonly encountered in mammalian systems is given in Table 3.4.
Two types of damage-response pathways exist: one is the repair pathway and the other is a tolerance mechanism (Friedberg, 1994). In repair mechanisms, the DNA damage is removed, while tolerance mechanisms circumvent the damage without fixing it. Tolerance mechanisms are by definition error-prone. Certain repair mechanisms reverse the DNA damage; for example, by removal of adducts from bases and insertion of bases into AP sites. One example of direct reversal is the removal of small alkyl groups from the O6 portion of guanine by alkyltransferases.
Alkyltransferases directly transfer the alkyl (methyl or ethyl) group from the DNA base guanine to a cysteine acceptor site in the alkyltransferase protein (Pegg and Byers, 1992). In microorgan- isms, the intracellular concentration of the alkyltransferase protein is regulated by environmental factors, including the presence of the alkylating agents themselves. A similar adaptation may occur in certain mammalian tissues in response to DNA-damaging agents as well as treatments
Figure 3.15 Schematic representation of chemical and radiation-induced lesions in DNA. (Adapted from Fry et al., 1982, with permission of the authors and publisher.)
causing an increase in cell proliferation. However, the increase in the alkyltransferase protein seen in mammalian tissues is much less than that in bacteria. In mammalian tissues, the level of the alkyltransferase protein is a major factor in the resistance of some cancer cells to certain chemotherapeutic agents (Chapter 20). Direct reversal of the premutational lesion by the alkyl- transferase reaction restores normal base pairing specificity.
Table 3.4 Types of DNA Repair
1. Direct reversal of DNA damage
2. Base excision repair
Glycosylase and AP endonuclease
3. Nucleotide excision repair T-T, C-C, C-T repair “Bulky” adduct repair
4. Double-strand break repair Homologous recombination (HR) Nonhomologous DNA end joining (NHEJ)
5. Mismatch repair
Repair of deamination of 5-Me cytosine
Repair of mismatches in DNA due to defective repair, etc.
Modified from Myles and Sancar, 1989, and from Lieber, 1998.
The excisional repair of DNA may involve either the removal of a single altered base hav- ing a relatively low-molecular-weight adduct, such as an ethyl or methyl group, and is termed base excision repair, or the repair may involve a base with a very large bulky group adducted to it (nucleotide excision repair). The linkage of two bases seen in the dimerization of pyrimidines by ultraviolet light is also repaired by the latter pathway. This nucleotide excision pathway is represented diagrammatically in Figure 3.16.
Nucleotide excision repair in multicellular organisms involves a series of reactions noted in the figure. These include recognition of the damage, unwinding of the DNA, 3′ and 5′ sequen- tial dual incisions of the damaged strand, repair synthesis of the eliminated patch, and final liga- tion. Each of these steps, as noted in the figure, involves a number of different proteins. In Table
3.5 may be seen a listing of the various proteins occurring in different fractions and their func- tions in the process of nucleotide excision repair (Petit and Sancar, 1999).
Other studies (cf. Sancar and Tang, 1993; Hanawalt, 1994) have also demonstrated that nucleotide excision repair in many instances occurs simultaneously with gene transcription. In fact, Hanawalt and associates showed earlier (cf. Bohr et al., 1987) that nucleotide excision re- pair occurred preferentially in genes that were actively being transcribed. For the final resynthe- sis of the segment of excised DNA, both the proliferating cell nuclear antigen (PCNA) as well as at least two different DNA polymerases (δ or ε) are needed to complete the repair process to- gether with a ligase (Sancar, 1994).
Since animal cell DNA polymerases are not absolutely faithful in their replication of the template strand, there is the potential for a mutation to occur in the form of one or more mis- paired bases during the process outlined above. This possibility is greater in the case of nucle- otide excision repair as compared to simple base excision since a much longer base sequence is removed and resynthesized during the nucleotide excision mechanism. The existence and ulti- mate characterization of a number of the proteins involved in nucleotide excision repair has been the result of human diseases in which defects in this mechanism are known. In particular, the disease xeroderma pigmentosum is an autosomal recessive (Chapter 5) condition in which most patients are highly sensitive to exposure to ultraviolet light. Thus, on chronic exposure to sun- light, such individuals have a much greater risk of developing skin cancer than normal individu- als. This fact emphasizes the potential importance of altered DNA repair in the development of neoplasia.
While the repair of adducts as indicated above involves several possible pathways, the re- pair of double DNA strand breaks is more complicated and as a result more prone to error than either the excisional or direct reversal pathways. Single-strand breaks may result from a variety of alterations by chemicals or radiation and, as noted above, during the repair process itself. Double-strand breaks in DNA are largely the result of ionizing radiation or high doses of alkylat- ing carcinogens such as nitrogen mustard or polycyclic hydrocarbons, although even under nor- mal conditions, transient double-strand DNA breaks occur as the result of the normal function of topoisomerases involved in the winding and unwinding of DNA and in antibody formation (Chapter 19).
In Figure 3.17 may be noted a schematic diagram of three forms of double-strand DNA repair. Recombinational repair or homologous recombination (HR) is more commonly seen in lower eukaryotes such as yeast while the nonhomologous end joining (NHEJ) pathway of double- strand DNA repair is more commonly seen in higher vertebrates (Van Dyck et al., 1999). The single-strand annealing pathway has not yet been well studied in higher vertebrates. While the exact mechanisms involved in each of these steps will not be considered in detail here, the inter- ested reader is referred to more detailed references (Pastink and Lohman, 1999; Lieber, 1998; Featherstone and Jackson, 1999). In general, in the HR and NHEJ pathways, specific proteins interact with the open ends of the DNA, members of the Rad52 group genes in the case of HR
Figure 3.16 Model for transcription-independent nucleotide excision repair of DNA in humans. (1) The damage is first recognized in an ATP-independent step by the short-lived XPA·RPA complex. In a second, ATP-dependent step, the damaged DNA-bound XPA·RPA complex recruits XPC and TFIIH, to form the preincision complex 1 (PIC1). TFIIH possesses both 3′-5′ and 5′-3′ helicase activities, respectively through its XPB and XPD subunits and unwinds DNA by about 20 base pairs around the damage. (2) XPG binds the PIC1 complex while the molecular matchmaker XPC dissociates, leading to the more stable PIC2 exci- nuclease complex. (3) PIC2 recruits XPF·ERCC1 (F-1) to form PIC3. XPG makes the 3′ incision and F-1 makes the 5′ incision a fraction of a second later, in a concerted but asynchronous mechanism. (4) The excised damaged fragment is released by the excinuclease complex, leaving in place a postincision com- plex whose exact composition is still unclear. The proliferating cell nuclear antigen (PCNA) forms a torus around the DNA molecule associating with DNA polymerase δ and/or ε [Pol ε (δ)] (Tsurimoto, 1998) and a DNA ligase replacing the postincision complex with these repair synthesis proteins. (5) The gap is filled and the repair patch is ligated. (From Petit and Sancar, 1999, with permission of authors and publisher.)
(Van Dyck et al., 1999) and the Ku70 and Ku80 proteins in the NHEJ pathway (Featherstone and Jackson, 1999). A DNA-dependent protein kinase (DNA-PKcs) as well as the protein interacting with the DNA ligase (XRCC4) is involved in this mechanism. It should be noted—as indicated in the legend to the figure—however, that these mechanisms are quite error-prone and only un- der the best of circumstances result in a faithful recapitulation of the normal DNA sequence.
Double-strand breaks may occur at sites of single-strand DNA resulting from adduction of bulky molecules, preventing further polymerase action and subsequent endonuclease cleavage and resulting in double-strand breaks and potential chromosomal aberrations (Kaufmann, 1989).
Incorrectly paired nucleotides may occur in DNA as a result of DNA polymerase infidel- ity, formation and/or repair of apurinic and nucleotide excision sites, double-strand DNA repair, and metabolic modification of specific bases. Mismatch repair can be distinguished from nucle- otide and base excision repair by several characteristics. Nucleotide and base excision repair generally involves the recognition of nucleotides/bases that have been chemically modified or fused to an adjacent nucleotide. In contrast, mismatch repair recognizes normal nucleotides which are either unpaired or paired with a noncomplementary nucleotide (cf. Fishel and Kolod- ner, 1995). Thus, mismatch repair may become involved in virtually any of the types of DNA repair seen in Table 3.4 with the possible exception of the direct reversal of DNA damage. The various combinations of gene products involved in several of the types of mismatch repair are seen in Figure 3.18. While the nomenclature of the various components varies depending on the phyla—e.g., eukaryotes, yeast, vertebrates—a functional similarity occurs throughout, most faithful in eukaryotes. As noted from the figure, recognition of the mismatch appears to be a major function of the MSH2 (hMSH2 in the human) while MSH3 and MSH6 are involved in the
Figure 3.17 Schematic representation of pathways involved in the repair of double-strand breaks in DNA. (a) The first step in recombinational repair is the formation of 3′ single-stranded tails by exonucle- olytic activity followed by invasion of a homologous undamaged donor sequence. Repair synthesis and branch migration lead to the formation of two Holliday junctions, i.e., a single DNA strand linking two double-stranded DNA molecules. Resolution of these intermediate structures results in the formation of two possible crossover and two possible noncrossover products (not shown). The fidelity of this repair is dependent on the exact complementation of the unaffected double-strand by the strands undergoing repair. (b) In the single-strand annealing pathway, exposures of regions of homology during resection of the 5′ ends allows formation of joint molecules. Repair of the double-strand break is completed by removal of nonhomologous ends and ligation. As a consequence, a deletion is introduced in the DNA. (c) Nonhomolo- gous end joining is based on religation of the two ends involving a complex of proteins, some of which are indicated in the figure and may involve the deletion and/or insertion of nucleotides. (Adapted from Pastink and Lohman, 1999, with permission of authors and publisher.)
specificity of binding itself (Fishel and Wilson, 1997). Thus, these complexes act as sensors of mismatch as well as other structural changes in the genome (Modrich, 1997; Li et al., 1996; cf. Fishel and Wilson, 1997). As in the case of other types of repair following the recognition and interaction with the mismatch repair proteins, the normal sequence is restored following removal of the mismatch DNA, resynthesis, and ligation (cf. Jiricny, 1998).
As an example of the importance of mismatch DNA repair, the extent of endogenous DNA damage and subsequent repair processes in normal human cells in vivo is seen in Table 3.6. With the possible exception of some single-strand break repair, all the other types of damage are those monitored by the mismatch repair mechanism and repaired under normal conditions. Obviously, a defect in this repair system may result in a dramatic increase in mutational events and in neo- plasia, as later discussions show (Chapter 5).