The role of endogenous and exogenous DNA damage and mutagenesis
Errol C Friedberg，Lisa D McDaniel and Roger A Schultz
Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas TX, 75390-9072, USA
Abstract
The field of DNA damage responsiveness in general, and the consequences of endogenous and exogenous base damage in DNA, in particular, has made new and exciting contributions to our increasing understanding of the initiation and progression of neoplasia in humans. This article presents some of the highlights in this area of investigation, with a particular emphasis on DNA repair, the tolerance of DNA damage and its contribution to mutagenesis, and DNA damage checkpoint regulation.
Abbreviations: AAF, acetylaminofluorene; APOBEC3G, apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G; ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3-related; ATRIP, ATR-interacting protein; CSA, Cockayne syndrome group A gene; CSN, COP9 signalosome; FA, Fanconi anemia; MAC, mutagenesis in aging colonies; MMC, mitomycin C; MMS, methylmethane sulfonate; NER, nucleotide excision repair; NHEJ, non-homologous end joining; ORF, open reading frame; RNAi, RNA interference; RPA, replication protein A; XP, xeroderma pigmentosum
Introduction
The role of both endogenous (spontaneous) and exogenous (environmental) DNA damage in the initiation and progression of neoplasms is unassailable. Genes that participate in various mechanisms that protect cells from the generation of mutations in somatic cells — except in selected physiological situations, such as the generation of mutations to promote variability in immunoglobulin genes — are now appropriately designated as tumor suppressor or gatekeeper genes [1.].
Cells have evolved multiple, and often apparently redundant, biological responses to DNA damage that are conveniently classified as either ‘DNA repair’ or ‘DNA damage tolerance’ [2.] ( Figure 1). As the name implies, DNA repair embraces mechanisms for the enzyme-catalyzed reversal of damage, the excision of base damage (including inappropriate bases such as uracil), as well as nucleotides that are incorrectly incorporated during DNA replication (Figure 1). In addition to base damage, our understanding of DNA repair now embraces the restoration of both single- and double-strand breaks in the genome [3. and 4.]. The tolerance of DNA damage involves several distinct cellular responses, by which the potentially lethal effects of arrested DNA replication by damaged bases are mitigated ( Figure 1).
Figure 1. Cellular responses to DNA damage. The response to DNA damage (yellow box) results in either tolerance or damage repair (blue boxes), with damage repair represented by a variety of specific damage repair pathways. These events are modulated and facilitated by cell cycle checkpoint mechanisms that arrest cell cycle progression at various points (orange box). The recent insights into the complexities of these events, as summarized in this review, are shown here to depict their newly discovered role(s) in these processes (green boxes).
Both the efficiency and kinetics of DNA repair and DNA damage tolerance are influenced by regulatory responses. With the advent of microarray technologies, we are just beginning to appreciate the magnitude, variation and significance of the extensive transcriptional responses to various types of genomic insult (Figure 1). Additionally, the role of DNA damage in the activation of cell cycle checkpoints is now a burgeoning field, involving multiple complex pathways that transduce signals from sites of DNA damage and altered DNA replication to repair and damage tolerance effector pathways ( Figure 1).
In this review, we highlight some recent advances in selected aspects of the plethora of biological responses to DNA damage, with a particular emphasis on mechanisms of mutagenesis from endogenous and exogenous damage.
New insights into DNA repair by the reversal of base damage
Several forms of base damage to DNA are repaired by biochemical reactions that directly reverse the damage, restoring affected bases to their native chemistry and conformation. Among the several genes that are required for the maintenance of genomic integrity in the face of alkylation damage to DNA in Escherichia coli is one called alkB, the specific function of which was unknown until very recently. In a model example of the utility of bioinformatics, Eugene Koonin and his colleagues [5.] have identified a domain in the translated alkB sequence that is suggestive of an -ketoglutarate- and Fe(II)-dependent dioxygenase. Two groups [6.?? and 7.??] have now independently shown that purified AlkB protein from E. coli repairs the cytotoxic lesions 1-methyladenine and 3-methylcytosine, by reversing such damage via a deoxygenase reaction that requires oxygen, -ketoglutarate and Fe(II). The bacterial alkB gene is conserved in eukaryotes, including human cells — in which there are two structural homologs, hABH2 and hABH3 — where it, presumably, subserves the same function.
More recent studies [8.] have demonstrated that the E. coli alkB and human hABH3 gene products also repair these lesions in RNA, extending the range of the repair of biological macromolecules to this class of polynucleotides. The repair of proteins [9.] and of deoxyribonucleoside triphosphate precursors of DNA [10.] has been documented previously.
New complexities in nucleotide excision repair
Nucleotide excision repair (NER) is a major form of repair for DNA base damage that results in distortions of DNA structure that (among other effects) interfere with normal base pairing. As such, it is suited to many forms of exogenous base damage, such as cyclobutane pyrimidine dimers induced by exposure to sunlight, a potent evolutionary driving force. NER encompasses the repair of both transcriptionally silent and transcriptionally active regions of the genome, by somewhat distinct mechanisms [11.]. These distinctions primarily center around the recognition of base damage. In particular, it is believed that damage recognition in transcriptionally active DNA is effected by arrest of the transcriptional machinery. One of the gene products required for transcription-coupled NER is the CSA protein, the product of the Cockayne syndrome group A gene (CSA) [11.].
A recent study [12.??] has identified human CSA protein in a multiprotein complex that includes the COP9 signalosome (CSN), a regulator of cullin-based ubiquitin ligases. This study also identified a second multiprotein complex, containing the COP signalosome, except that instead of CSA protein, this complex contains another protein that is required for NER, DDB2, a 48 kDa protein, which, together with DDB1 protein, comprises a heterodimer that is involved in DNA damage recognition during transcriptionally independent NER [12.??]. Mutations in the DDB2 gene have been identified in several individuals with the NER-defective and skin-cancer-prone hereditary disease xeroderma pigmentosum (XP), belonging to genetic complementation group E [13. and 14.]. The authors suggest that, following exposure to UV radiation, the DDB2 complex binds to chromatin and the COP signalosome dissociates and activates ubiquitin ligase E3 activity. By contrast, when the transcriptional machinery is arrested by UV radiation induced base damage, the CSA complex recruits the COP signalosome and inactivates the ubiquitin ligase [12.??].
Something new in mismatch repair
One of the central debates about neoplastic transformation concerns the strong mutator phenotype of neoplasms, which cannot be accounted for by the multiplicative sum of the spontaneous mutation frequency in individual genes. An in-depth coverage of this topic is outside the scope of this review; however, among the possible explanations for this strong mutator phenotype is the notion that genes involved in mismatch repair of DNA become inactivated by one or another genetic and/or epigenetic mechanisms [15. and 16.], and that the ensuing mutator phenotype increases the probability (by random or possibly non-random mechanisms) of inactivation of other DNA repair genes. In support of this notion, a recent study examined clones of cells with either an inactive or active hMSH6 gene [17.] in which expression of hMLH1 was silenced by promoter hypermethylation. The additional inactivation of this gene in cells mutant for hMSH6 resulted in a higher mutation rate and a different mutational spectrum than cells that are wild-type for hMSH6 [17.].
Most mutations that are associated with defective mismatch repair are the result of inactivation of genes for this DNA repair process. A recent study, however, has demonstrated that some exogenous mutagens can inactivate mismatch repair proteins directly [18.?]. Specifically, chronic exposure of yeast to cadmium inactivates mismatch repair in vivo and this effect can be reproduced in vitro. This is the first clear demonstration of an exogenous agent promoting genomic instability through direct effects on ‘guardians of the genome’, rather than on the genome itself, and begs more extensive examination of this mechanism of environmentally induced genomic instability.
What’s new in strand-break repair?
Our understanding of the details of DNA-strand-break repair, particularly that in mammalian cells, has, until recently, lagged behind that of the repair of base damage: however, the past 4–5 years have witnessed impressive progress in this area. We now know that double-strand breaks can be repaired by either homologous recombination by a variety of different mechanisms, or by the direct fusion of broken ends (non-homologous end joining [NHEJ]), an area that has significant overlap with V(D)J recombination in the immune system. A newly discovered gene called Artemis has been shown to be involved in V(D)J recombination in the immune system [19.]. More recently, it was demonstrated that the Artemis protein is a single-strand-specific exonuclease. It also complexes with the DNA-dependent protein kinase (DNA-PKCS), an integral component of the NHEJ machinery [20.??]. Complex formation is followed by phosphorylation of Artemis by DNA-PKCS,, an event that converts it to an endonuclease that can open DNA hairpins that are generated during V(D)J recombination [20.??].
Defects in NHEJ in the immune system accelerate the formation of lymphomas in mice. However, this process of strand-break rejoining can also suppress tumors in cells that do not undergo V(D)J recombination. A recent study has demonstrated that haploinsufficiency for the Lig4 gene (which encodes DNA ligase IV) results in the development of non-lymphomatous tumors in a cancer-prone mouse strain [21.]. Hence, even a modest reduction in NHEJ activity promotes tumorigenesis in mice.
DNA damage tolerance: the role(s) of error-prone DNA polymerases
Another area of significant progress has emerged from the discovery of a large repertoire of DNA polymerases (especially in mammalian cells), endowed with the ability to bypass many types of spontaneous and exogenously generated forms of base damage, often (but not always) leading to mutations [22.]. In E. coli, one of these polymerases, called Pol IV and encoded by the dinB gene, has been implicated in spontaneous mutagenesis [23.?]. Spontaneous mutagenesis can occur in rapidly growing and in stationary phase E. coli by different processes. There has been conflicting data as to the requirement for Pol IV for the latter process. A recent study indicates that this confusion apparently originates from the type of mutant strain used. The dinB gene is part of a four-gene operon with three downstream genes of unknown function. Hence, some mutations in the dinB gene can result in polar effects. Non-polar mutants do not result in spontaneous mutations in rapidly growing cells [23.?].
It is believed that when replication is blocked by DNA damage in a strand (either leading or lagging), polymerase switching transpires, enabling the bypass polymerase(s) to transiently occupy the primer template for replicative bypass and then to reoccupy this site when translesion synthesis is completed. If the replicative machinery is physically displaced from the primer-template during this process, replication of both DNA strands might be expected to arrest. An in vivo system was established in E. coli to address this question [24.??]. It was observed that an acetylaminofluorene (AAF) lesion in the leading strand did not affect the kinetics of lagging strand replication and vice versa. Hence, whatever the nature of the polymerase switching events during replicative bypass of base damage (translesion DNA synthesis), the replicative machinery does not appear to be completely disengaged from the arrested fork.
Tuberculosis is a pulmonary infection that is sometimes prone to lethal antibiotic resistance. A recent study has demonstrated that when Mycobacterium tuberculosis is exposed to various types of DNA base damage, a gene called dnaE2 (believed to encode a novel DNA polymerase) is upregulated and results in an increased mutation frequency in the bacterium. It is suggested that mutations associated with spontaneous DNA damage might form the basis of the antibiotic resistance that is manifested by this organism [25.?].
Other aspects of spontaneous mutagenesis
It is well established that, in laboratory-derived strains of E. coli, mutagenesis can be promoted by stress conditions. However, the general evolutionary significance of this phenomenon has been questioned because laboratory strains are not representative of strains in the wild, growing in different natural ecological niches. A recent study collected nearly 800 E. coli isolates from around the world and examined mutagenesis in aging colonies (MAC) by exposure to starvation after a period of exponential growth [26.?]. Most natural isolates exhibited increased MAC. Although the nature of the mutagenesis was characteristic for each strain — the particular ecological niche from which the strain was isolated being a major determinant of the mutator phenotype — the study supports the notion that adaptive mutagenesis associated with stress-induced mutations is a general evolutionary strategy in E. coli.
The high spontaneous mutation of HIV is a principal scourge of the disease AIDS, but the primary mechanism(s) of this mutagenesis remains to be established. No less than three recent studies [27.?, 28.? and 29.?] have demonstrated that APOBEC3G (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G), an endogenous inhibitor of HIV-1 replication, is a cytidine deaminase that generates G→A mutations in the viral DNA, presumably by converting cytidine to uracil in the viral DNA minus strand, thereby promoting the formation of U:A base pairs. Whereas this hypermutation is considered to have evolved as a viral defense mechanism leading to inactivation of the virus, the accumulation of APOBEC3G-induced non-lethal mutations could potentially promote variation in primate lentiviral populations, including HIV.
Checkpoint control and initiating signals for responses to DNA damage
ATR (ataxia telangiectasia and Rad3-related) and ATM (ataxia telangiectasia mutated) are kinases that play central roles in responses to various types of DNA damage, notably that produced by ionizing radiation [30. and 31.]. ATR is known to phosphorylate substrates such as Brca1, Chk1 protein, p53 and Rad17. These phosphorylated substrates, in turn, mediate inhibition of DNA replication and progression through the cell cycle and promote DNA repair and other effector responses [30. and 31.]. Many DNA-damaging agents can elicit the ATR-mediated DNA damage response, therefore an issue of considerable interest is whether different sensors function for different types of damage, or whether all are processed to a common intermediate. A recent study has demonstrated that RPA (replication protein A; a single-stranded DNA binding protein) stimulates the binding of the ATR–ATRIP (ATR-interacting protein) complex to single-stranded DNA, stimulating the phosphorylation of Rad17 protein that is bound to DNA [32.??]. These studies suggest that single-stranded DNA coated with RPA is the key denominator of varying types of DNA damage that recruits ATR–ATRIP and initiates the DNA-damage-signaling cascade.
It has also been proposed that the process of retroviral DNA integration is ‘sensed’ as DNA damage by host cells. In an independent study, it was demonstrated that the ATR kinase (but not the ATM kinase) is required for the successful integration of retroviral RNA [33.?].
New genes for biological responses to DNA damage?
The genomics era has facilitated the identification of new genes that are involved in biological responses to DNA damage. In addition to expression-array studies, other techniques have been employed in attempts to gain such insights. One recent study identified six novel genes that are involved in biological responses to UV radiation or methylmethane sulfonate (MMS) by screening a collection of >2800 yeast-deletion mutants. In each mutant, a single ORF was replaced by a cassette, containing two unique sequence tags, thus allowing for their identification by hybridization to a high-density oligonucleotide array [34.?].
Another study used RNAi to uncover genes in the nematode worm Caenorhabditis elegans. A total of 61 genes were found to affect genomic stability in somatic cells and spontaneous mutagenesis in the germ line [35.?]. Many of the genes uncovered are novel ORFs with no known function.
Fanconi anemia and BRCA
Fanconi anemia (FA) is a clinically heterogeneous human disorder that is associated with genomic instability, cancer predisposition and cellular sensitivity to certain DNA damaging agents, including mitomycin C (MMC). The disease is genetically complex, represented by eight genetic complementation groups. Genes representing six of the FA complementation groups had been previously cloned and elucidation of the function of the products of these genes over the last decade has provided insight into a previously unrecognized regulatory pathway for DNA-damage response. Five of these (groups FA-A, -C, -E, -F and -G) were shown to participate in the formation of a complex that directs the monoubiquitination of the product of the gene mutated in a sixth group (FA-D2). Moreover, it was recognized that this monoubiquitination leads to the targeting of the FANCD2 protein to BRCA1 nuclear foci and subsequent signaling through BRCA2 and RAD51 for the repair of DNA damage. RAD51 is a crucial component of homologous recombination and this, therefore, suggests that the FA complex mediates error-free homologous recombination during S-phase arrest. These results are consistent with studies that have reported an increased mutation frequency in FA cells, relative to normal controls and, more specifically, an increase in the occurrence of deletion mutations. What remained a mystery here was which genes were mutated in the other FA groups (FA-B and -D1) and how the protein products of these two unidentified genes participated in the same DNA-damage-signaling pathway.
A recent report by Howlett et al. [36.??] demonstrates biallelic mutations in the BRCA2 gene in both of these complementation groups. The findings illustrate that all eight of the FA gene products are involved in a single pathway that mediates DNA repair in response to DNA damage caused by agents such as MMC and diepoxybutane, which are both known to produce crosslinks in DNA. The MMC sensitivity of FA-D1 fibroblast was complemented, following expression of the wild-type BRCA2 protein. The fact that two distinct complementation groups are defined by mutations in a single gene suggests interallelic complementation or dominant activity for certain mutant alleles.
Conclusions
The multiple mechanisms by which both natural (spontaneously-derived) and exogenous (environmentally-derived) mutations arise in cells and, hence, can trigger neoplasia are becoming clearer. Mutagenesis is a universal fact of life: the genetic diversity it generates in germ cells is essential for Darwinian evolution. From an evolutionary point of view, there is no intrinsic reason to suppress mutations in somatic cells, provided that they do not result in deleterious phenotypes before reproductive age. However, diseases such as XP, in which a hereditary defect in a specific DNA repair modality predisposes the individual to lethal multiple skin cancers well before the onset of puberty, provide dramatic evidence that preventing an excessive mutational burden in somatic cells is as essential to life as the generation of a threshold of mutations in the germ line. Biological evolution has apparently achieved a delicate balance in which mutations are tolerated at certain levels in both germ line and somatic cells. The preponderance of the evidence indicates that mutations are stochastic events in genes. Hence, should they transpire in genes that are crucial for normal cellular proliferation, the price might be neoplasia. As there is no a priori reason to select against neoplasia in organisms past their reproductive life, there is no reason to expect selection for anti-mutagenic mechanisms that specifically favor oncogenes and tumor suppressor genes. Nor is there evidence of such mechanisms.
Meanwhile, our understanding of the multiple ways in which cells are subjected to genomic insult and the diverse responses to such damage continues to expand. Such responses appear, primarily, to be designed to avoid cell death, not mutations. Indeed, in some cases, mutations are imposed on cells as a strategy to avoid death.
Acknowledgements
We apologize to the many authors of outstanding papers that were not included here due to space limitations and the general scope of this issue.
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