- Email: [email protected]
Special issue on induced tandem repeat instability
Mutation Research 598 (2006) 1–5
Editorial
Special issue on induced tandem repeat instability The measurement and detection of spontaneous and induced mutations is rendered extremely difficult by the low frequency of these events in somatic and germ cells. In 1993, a seminal article by Dubrova et al. [1] examined the relationship between tandem repeat (TR) DNA mutation and exposure of male mice to ionizing radiation. Because TR DNA sequences (in this case minisatellite DNA [2]) show much higher rates of mutation than unique sequence DNA, the authors hypothesized that these regions may be sensitive to mutagens and permit the direct observation of induced mutation arising in the germ cells of irradiated mice. In support of this hypothesis, the authors observed a significant increase in TR mutation in the offspring of male mice exposed to ionizing radiation, providing a new and sensitive approach to detect induced mutation and genomic instability. The study measured induced mutation at low doses of radiation using at least an order of magnitude fewer animals than other assays (e.g., specific locus test), yet obtained similar estimates for doubling doses compared to other, more traditional, germ cell assays [3]. Remarkably, increased rates of TR mutation have since been observed to result from environmental exposures in: (1) humans, birds and plants sampled from sites of radioactive contamination [4–9], (2) plants sampled in metal contaminated areas [10] and (3) gulls [11,12] and mice [13,14] inhabiting contaminated industrialized locations. However, nearly as many studies have been unable to measure induced mutation in TR regions of highly exposed organisms [15–22], and therefore the relationship between germline TR instability and exposure to toxic chemicals remains contentious. Today, new technologies using single-molecule and small-pool PCR permit the direct observation of both somatic and germline mutation at virtually any type of TR sequence in any tissue. The 12 reviews and original research contributions in this special issue of Mutation Research provide an overview of our current understanding of TR DNA instability and
its application in the measurement of induced mutation. These articles focus on repeat sequence instability in non-cancer tissues (i.e., they do not cover microsatellite instability in carcinomas that are the result of defects in mismatch repair), and address both germline and somatic instability. Despite over a decade of research, the mechanisms and relevance of TR instability have remained controversial. Germline expansion in a number of specific TR regions is directly causative of disease and has obvious clinical significance (e.g., the FMR1 and myotonic dystrophy type 1 microsatellites). The importance of repeat instability in muscular and neurodegenerative disorders (e.g., Huntington disease, Fragile X syndrome, spinocerebellar ataxias, etc.) has recently been the focus of a Special Issue in Nature Reviews Genetics (October 2005, vol. 6, no. 10), highlighting the timeliness and importance of the topic. However, the vast majority of TR loci are found in non-coding regions of the genome that have no known function (‘junk DNA’). Therefore, the functional significance of induced mutation at TRs is uncertain. Furthermore, the mechanisms that result in induced instability at the various types of TR sequences are currently unclear. For some loci it has been demonstrated that induced mutation results from ‘indirect’ effects manifested elsewhere in the genome, and that the consequences of these effects can be stably transmitted to offspring through multiple generations [23,24]. Indirect mutation, and epigenetic transmission of genetic instability to non-exposed descendants, starkly contrast traditional paradigms in genetic toxicology [25]. Lastly, the relationship or correlation between mutation in TR sequences and unique sequence DNA remains a mystery. Although there are many unanswered questions, the application of TR mutation arguably remains the only assay capable of detecting statistically significant increases in germline mutation resulting from exposure to environmental levels of mutagens. Consequently, it
0027-5107/$ – see front matter. Crown Copyright © 2006 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2006.01.006
2
Editorial / Mutation Research 598 (2006) 1–5
stands to reason that a great deal of research emphasis should be placed on: (a) deciphering the mechanisms involved in both spontaneous and induced TR mutation and (b) increasing our understanding of the biological significance of non-functional TR sequence mutation. Furthermore, it is also important to consider that ‘junk DNA’ may have some functional significance (e.g., [26]) and that repetitive DNA may play an important role in the rapid evolution of species [27]. The majority of articles in this special issue of Mutation Research provide an overview of our current understanding of the utility of TR mutation in measuring induced genetic instability, the importance and relevance of TR DNA mutation assays, and the potential mechanisms involved in generating instability across the different repeat types. An introductory commentary by Armour [28] argues the importance of TR DNA, its utility for the study of mutation, and describes the functionality of a subset of TR DNAs. Armour establishes TR regions as effective reporters of genetic instability and induced mutation, and discusses the functional repercussions of these mutations. Gomes-Pereira and Monckton provide a review of instability at microsatellite repeats associated with human genetic disorders [29]. In addition to germline expansion, the authors highlight some exciting new data on chemical modifiers of somatic repeat expansion and suggest that these chemical treatments could potentially be useful as therapeutic tools to modulate disease progression over time. Somers [30] reviews induced expanded simple tandem repeat (ESTR; [31,32]) mutation in the male mouse germline. His review details the discrepancies in the field, including controversy over the timing of induced mutation, and the types and efficiencies of DNA lesions that cause destabilization. The non-targeted nature of induced mutation is discussed in the context of the mouse models that have been used to examine instability. It is clear that the mechanisms that result in ESTR destabilization are unknown and much more work is needed. Barber and Dubrova [33] present data over-viewing radiation-induced genomic instability in the context of trans-generational effects. Work from this pioneering laboratory was the first to show that radiation-induced germline instability can persist through multiple generations. Radiation-induced instability arising in nonexposed progeny poses a fundamental challenge to the way radiation risk assessment is currently undertaken, and suggests that the risks of exposure to ionizing radiation could be greater than previously predicted. It is clear from their review that the molecular and cellular events that contribute to the manifestation of persistent instability are unknown and require further research. Work
from Niwa’s laboratory in the early 1990’s was also ground-breaking in establishing the important potential role of TR in understanding induced genetic instability. Niwa’s review [34] presents further hypotheses to address discrepancies in the timing of induced TR mutation. Niwa argues that mutations arise in the zygote as a result of inheritance of damaged paternal DNA, when DNA is damaged just prior to conception. This hypothesis is supported by his finding of increased mutation at both ESTR loci and the pink-eyed unstable locus of maternal alleles in descendants of irradiated fathers. The delayed effects observed in Niwa’s experiments demonstrate persistent instability, consistent with an epigenetic mechanism, and potentially similar to the mechanisms operating in trans-generational instability. Niwa effectively argues for a possible role of p53 in this response. Clearly more work is needed to decipher the mechanisms operating in both immediate and delayed mutation at TR regions. Cell culture assays facilitate dissecting the mechanistic aspects of repeat mutation. A cell culture assay is used to investigate TR mutation in an original contribution from Polyzos et al. [35]. The authors used a single-molecule PCR approach to measure induced ESTR instability at the Ms6-hm locus after exposure of cultured murine embryonic fibroblast cells to low doses of chemicals with different modes of action. At subtoxic doses of chemicals the authors find that a DNA alkylating agent (N-nitroso-N-ethylurea), a bulky DNA adduct forming agent (benzo(a)pyrene), and a topoisomerase inhibitor (etoposide) caused similar induction of ESTR instability in embryonic fibroblasts. In contrast, a protein kinase inhibitor (okadaic acid) did not cause significant ESTR mutation. The work takes us a step closer to understanding the types of lesions that can result in TR destabilization, and future work will examine a wider spectrum of chemicals. The development of a green fluorescent protein assay using flow cytometry to measure frameshift mutations at TR regions in mouse cell cultures is presented by Healy et al. [36]. The assay not only allows a detailed dose–response investigation of the chemicals that can result in destabilization at repeat units, but also allows the investigation of sequence specificity in the context of instability. Interestingly, treatment with model chemical mutagens or tumour promoters induced statistically significant sequence- and exposuredependent responses. The development of this assay will allow for more detailed mechanistic studies with a broad range of constructs and a variety of chemicals, to provide insight into mechanisms operating in repeat instability. In vitro work described by Wang and Vasquez [37] and Nakagama et al. [38] examines structural conforma-
Editorial / Mutation Research 598 (2006) 1–5
tions that repeat sequences may form that contribute to their instability, and the mechanisms potentially operating in repeat dynamics. Wang and Vasquez provide a review of the role of non-B DNA conformations in repeat mutation. Repetitive DNA elements are more prone to forming such structures, including hairpins, left-handed Z-DNA and intramolecular triplexes, which can lead to both expansion and contraction in allele size. The authors discuss potential mechanisms operating in mutation, including the involvement of mismatch repair proteins, nucleotide excision repair proteins, topoisomerases and structure-specific nucleases. Nakagama et al. [38] examine the molecular mechanisms involved in the maintenance of stability at G-rich repetitive sequences, such as minisatellites and ESTRs. G-rich DNA sequences can adopt a variety of non-canonical DNA structures, including G4 DNA. These sequences can be recombination and mutation hotspots in vivo, and may have the capacity to block the progression of DNA polymerases. The RNA–protein complexes, hnRNP A1 and hnRNP A3, bind to these sequences and can unwind the secondary structures. Therefore, these proteins might facilitate DNA replication at these loci and assist in the maintenance of genomic stability. Reviews by Cederberg and Rannug [39] and by Bichara et al. [40] examine model organisms to dissect the mechanisms operating in TR mutation. Cederberg and Rannug review work examining human minisatellites integrated into the Saccharomyces cerevisiae genome. Both human and yeast minisatellite mutation is driven by meiotic recombination, but the structural changes that are generated by the recombination events differ. In contrast to human minisatellites, inter-allelic transfer of repeats in yeast occurs by conversion, and not crossing over, and several chromatids can be involved in successive recombination events in one meiosis. Intrinsic properties of the minisatellite do not govern instability in meiosis, but do play a role in instability arising in mitosis. The results demonstrate that meiotic mutation in the integrated human minisatellites is highly dependent on genomic location and that a number of genes involved in initiation of recombination and DNA repair are required for mutation induction and maintenance of stability. Bichara et al. [40] review and present new research findings on microsatellite instability in prokaryotes, with emphasis on studies using the model organism Escherichia coli. Both the frequency and types of mutations that occur in E. coli are influenced by the DNA sequence of the repeat, the number of repeat units, and the cellular pathways that process the TR sequences. DNA strand slippage is the general model presented to explain prokaryotic TR instability, with misaligned
3
DNA sequences stabilized by favorable base pairing of complementary sequences and by the predisposition of TRs to form relatively stable secondary structures. The work reviews the cellular processes that are potentially involved, including replication, recombination and a variety of DNA repair pathways, and presents new experimental data suggesting that translesion synthesis polymerases (PolII, PolIV and PolV) do not contribute significantly to TR instability in E. coli. The last review in the issue, by Singer et al. [41], examines traditional mouse germ cell mutation assays and their correlation to TR instability and transgenic rodent (TGR) assays. The latter two assays offer considerable advantages over the traditional approaches. An understanding of the correlation among assays, and the relationship between repeat mutation and other types of DNA damage and mutation, is crucial in order to decipher the biological implications of repeat instability. This detailed comparative study examined data in the literature pertaining to the mutagenicity of chemicals and radiation in the dominant lethal assay, the morphological specific locus test, the heritable translocation assay, ESTR and/or TGR mutation assays. Only a handful of mutagens have been examined using several of the methods and the results of the paper demonstrate that development of the TGR and ESTR germline mutation assays for regulatory testing will require more robust and extensive characterization of assay performance. The laboratories represented in this special issue have made important contributions in this field and I am extremely grateful to them for taking the time to contribute. The high quality of the articles is the result of the great care put into the preparation of the manuscripts in addition to the helpful suggestions provided by the referees. I would like to take the opportunity to thank all of the authors and reviewers for what I hope will be a highly regarded review on the status of TR mutation in the context of induced instability. Finally, I would like to express my gratitude to Peter Stambrook and Elsevier for allowing me to act as guest editor. In addition to having been a fantastic learning experience, this has been an invaluable opportunity to bring together opinions and findings from complementary laboratories in what I view as an exciting, dynamic and important research field. References [1] Y.E. Dubrova, A.J. Jeffreys, A.M. Malashenko, Mouse minisatellite mutations induced by ionizing radiation, Nat. Genet. 5 (1993) 92–94. [2] A.J. Jeffreys, V. Wilson, S.L. Thein, Hypervariable ’minisatellite’ regions in human DNA, Nature 314 (1985) 67–73.
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Editorial / Mutation Research 598 (2006) 1–5
[3] Y.E. Dubrova, M. Plumb, J. Brown, J. Fennelly, P. Bois, D. Goodhead, A.J. Jeffreys, Stage specificity, dose response, and doubling dose for mouse minisatellite germ-line mutation induced by acute radiation, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 6251– 6255. [4] Y.E. Dubrova, V.N. Nesterov, N.G. Krouchinsky, V.A. Ostapenko, R. Neumann, D.L. Neil, A.J. Jeffreys, Human minisatellite mutation rate after the Chernobyl accident, Nature 380 (1996) 683–686. [5] Y.E. Dubrova, V.N. Nesterov, N.G. Krouchinsky, V.A. Ostapenko, G. Vergnaud, F. Giraudeau, J. Buard, A.J. Jeffreys, Further evidence for elevated human minisatellite mutation rate in Belarus eight years after the Chernobyl accident, Mutat. Res. 381 (1997) 267–278. [6] Y.E. Dubrova, R.I. Bersimbaev, L.B. Djansugurova, M.K. Tankimanova, Z. Mamyrbaeva, R. Mustonen, C. Lindholm, M. Hulten, S. Salomaa, Nuclear weapons tests and human germline mutation rate, Science 295 (2002) 1037. [7] Y.E. Dubrova, G. Grant, A.A. Chumak, V.A. Stezhka, A.N. Karakasian, Elevated minisatellite mutation rate in the postchernobyl families from ukraine, Am. J. Hum. Genet. 71 (2002) 801–809. [8] H. Ellegren, G. Lindgren, C.R. Primmer, A.P. Moller, Fitness loss and germline mutations in barn swallows breeding in Chernobyl, Nature 389 (1997) 593–596. [9] O. Kovalchuk, Y.E. Dubrova, A. Arkhipov, B. Hohn, I. Kovalchuk, Wheat mutation rate after Chernobyl, Nature 407 (2000) 583–584. [10] S.H. Rogstad, B. Keane, M.H. Collier, Minisatellite DNA mutation rate in dandelions increases with leaf-tissue concentrations of Cr, Fe, Mn, and Ni, Environ. Toxicol. Chem. 22 (2003) 2093–2099. [11] C.L. Yauk, J.S. Quinn, Multilocus DNA fingerprinting reveals high rate of heritable genetic mutation in herring gulls nesting in an industrialized urban site, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 12137–12141. [12] C.L. Yauk, G.A. Fox, B.E. McCarry, J.S. Quinn, Induced minisatellite germline mutations in herring gulls (Larus argentatus) living near steel mills, Mutat. Res. 452 (2000) 211–218. [13] C.M. Somers, B.E. McCarry, F. Malek, J.S. Quinn, Reduction of particulate air pollution lowers the risk of heritable mutations in mice, Science 304 (2004) 1008–1010. [14] C.M. Somers, C.L. Yauk, P.A. White, C.L. Parfett, J.S. Quinn, Air pollution induces heritable DNA mutations, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 15904–15907. [15] C.A. May, K. Tamaki, R. Neumann, G. Wilson, G. Zagars, A. Pollack, Y.E. Dubrova, A.J. Jeffreys, M.L. Meistrich, Minisatellite mutation frequency in human sperm following radiotherapy, Mutat. Res. 453 (2000) 67–75. [16] J.A. Armour, M.H. Brinkworth, A. Kamischke, Direct analysis by small-pool PCR of MS205 minisatellite mutation rates in sperm after mutagenic therapies, Mutat. Res. 445 (1999) 73–80. [17] A. Kiuru, A. Auvinen, M. Luokkamaki, K. Makkonen, T. Veidebaum, M. Tekkel, M. Rahu, T. Hakulinen, K. Servomaa, T. Rytomaa, R. Mustonen, Hereditary minisatellite mutations among the offspring of Estonian Chernobyl cleanup workers, Radiat. Res. 159 (2003) 651–655. [18] L.A. Livshits, S.G. Malyarchuk, S.A. Kravchenko, G.H. Matsuka, E.M. Lukyanova, Y.G. Antipkin, L.P. Arabskaya, E. Petit, F. Giraudeau, P. Gourmelon, G. Vergnaud, B. Le Guen, Children of chernobyl cleanup workers do not show elevated rates of mutations in minisatellite alleles, Radiat. Res. 155 (2001) 74–80.
[19] C. Satoh, N. Takahashi, J. Asakawa, M. Kodaira, R. Kuick, S.M. Hanash, J.V. Neel, Genetic analysis of children of atomic bomb survivors, Environ. Health Perspect. 104 (Suppl. 3) (1996) 511–519. [20] M. Kodaira, S. Izumi, N. Takahashi, N. Nakamura, No evidence of radiation effect on mutation rates at hypervariable minisatellite loci in the germ cells of atomic bomb survivors, Radiat. Res. 162 (2004) 350–356. [21] M. Stapleton, P.O. Dunn, J. McCarty, A. Secord, L.A. Whittingham, Polychlorinated biphenyl contamination and minisatellite DNA mutation rates of tree swallows, Environ. Toxicol. Chem. 20 (2001) 2263–2267. [22] N. Zheng, D.G. Monckton, G. Wilson, F. Hagemeister, R. Chakraborty, T.H. Connor, M.J. Siciliano, M.L. Meistrich, Frequency of minisatellite repeat number changes at the MS205 locus in human sperm before and after cancer chemotherapy, Environ. Mol. Mutagen. 36 (2000) 134–145. [23] Y.E. Dubrova, M. Plumb, B. Gutierrez, E. Boulton, A.J. Jeffreys, Transgenerational mutation by radiation, Nature 405 (2000) 37. [24] R. Barber, M.A. Plumb, E. Boulton, I. Roux, Y.E. Dubrova, Elevated mutation rates in the germ line of first- and secondgeneration offspring of irradiated male mice, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 6877–6882. [25] B.A. Bridges, Strange goings-on in the mouse germ line, DNA Repair (Amst.) 2 (2003) 1269–1272. [26] P. Andolfatto, Adaptive evolution of non-coding DNA in Drosophila, Nature 437 (2005) 1149–1152. [27] J.W. Fondon III, H.R. Garner, Molecular origins of rapid and continuous morphological evolution, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 18058–18063. [28] J.A.L. Armour, Tandemly repeated DNA: why should anyone care? Mutat Res. 598 (2006) 6–14. [29] M. Gomes-Pereira, D.G. Monckton, Chemical modifiers of unstable expanded simple sequence repeats: what goes up, could come down, Mutat. Res. 598 (2006) 15–34. [30] C.M. Somers, Expanded simple tandem repeat (ESTR) mutation induction in the male germline: lesssons learned from lab mice, Mutat. Res. 598 (2006) 35–49. [31] R. Kelly, M. Gibbs, A. Collick, A.J. Jeffreys, Spontaneous mutation at the hypervariable mouse minisatellite locus Ms6-hm: flanking DNA sequence and analysis of germline and early somatic mutation events, Proc. Biol. Sci. 245 (1991) 235– 245. [32] P. Bois, J. Williamson, J. Brown, Y.E. Dubrova, A.J. Jeffreys, A novel unstable mouse VNTR family expanded from SINE B1 elements, Genomics 49 (1998) 122–128. [33] R.C. Barber, Y.E. Dubrova, The offspring of irradiated parents, are they stable? Mutat Res. 598 (2006) 50–60. [34] O. Niwa, Indirect mechanisms of genomic instability and the biological significance of mutations at tandem repeat loci, Mutat. Res. 598 (2006) 61–72. [35] A. Polyzos, C. Parfett, C. Healy, G.R. Douglas, C.L. Yauk, Instability of expanded simple tandem repeats is induced in cell culture by a variety of agents: N-nitroso-N-ethylurea, benzo(a)pyrene, etoposide and okadaic acid, Mutat. Res. 598 (2006) 73–84. [36] C. Healy, M. Wade, A. McMahon, A. Williams, D. Johnson, C. Parfett, Flow cytometric detection of tandem repeat mutations induced by various chemical classes, Mutat. Res. 598 (2006) 85–102. [37] G. Wang, K.M. Vasquez, Non-B DNA structure-induced genetic instability, Mutat. Res. 598 (2006) 103–119.
Editorial / Mutation Research 598 (2006) 1–5 [38] H. Nakagama, K. Higuchi, E. Tanaka, N. Tsuchiya, K. Nakashima, M. Katahira, H. Fukuda, Molecular mechanisms for maintenance of G-rich short tandem repeats, Mutat. Res. 598 (2006) 120–131. [39] H. Cederberg, U. Rannug, Mechanisms of human minisatellite mutation in yeast, Mutat. Res. 598 (2006) 132–143. [40] M. Bichara, J. Wagner, I.B. Lambert, Mechanisms of Tandem Repeat Instability in Bacteria, Mutat. Res. 598 (2006) 144–163. [41] T.M. Singer, I.B. Lambert, A. Williams, G.R. Douglas, C.L. Yauk, Detection of induced male germline mutation: correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeats, Mutat. Res. 598 (2006) 164–193.
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Carole L. Yauk ∗ Environmental and Occupational Toxicology Division, Health Canada, Environmental Health Centre, Tunney’s Pasture, 0803A, Ottawa, Ont., Canada K1A 0L2 ∗ Tel.:
+1 613 941 7376; fax: +1 613 941 8530. E-mail address: Carole [email protected] Available online 14 February 2006
Editorial
Special issue on induced tandem repeat instability The measurement and detection of spontaneous and induced mutations is rendered extremely difficult by the low frequency of these events in somatic and germ cells. In 1993, a seminal article by Dubrova et al. [1] examined the relationship between tandem repeat (TR) DNA mutation and exposure of male mice to ionizing radiation. Because TR DNA sequences (in this case minisatellite DNA [2]) show much higher rates of mutation than unique sequence DNA, the authors hypothesized that these regions may be sensitive to mutagens and permit the direct observation of induced mutation arising in the germ cells of irradiated mice. In support of this hypothesis, the authors observed a significant increase in TR mutation in the offspring of male mice exposed to ionizing radiation, providing a new and sensitive approach to detect induced mutation and genomic instability. The study measured induced mutation at low doses of radiation using at least an order of magnitude fewer animals than other assays (e.g., specific locus test), yet obtained similar estimates for doubling doses compared to other, more traditional, germ cell assays [3]. Remarkably, increased rates of TR mutation have since been observed to result from environmental exposures in: (1) humans, birds and plants sampled from sites of radioactive contamination [4–9], (2) plants sampled in metal contaminated areas [10] and (3) gulls [11,12] and mice [13,14] inhabiting contaminated industrialized locations. However, nearly as many studies have been unable to measure induced mutation in TR regions of highly exposed organisms [15–22], and therefore the relationship between germline TR instability and exposure to toxic chemicals remains contentious. Today, new technologies using single-molecule and small-pool PCR permit the direct observation of both somatic and germline mutation at virtually any type of TR sequence in any tissue. The 12 reviews and original research contributions in this special issue of Mutation Research provide an overview of our current understanding of TR DNA instability and
its application in the measurement of induced mutation. These articles focus on repeat sequence instability in non-cancer tissues (i.e., they do not cover microsatellite instability in carcinomas that are the result of defects in mismatch repair), and address both germline and somatic instability. Despite over a decade of research, the mechanisms and relevance of TR instability have remained controversial. Germline expansion in a number of specific TR regions is directly causative of disease and has obvious clinical significance (e.g., the FMR1 and myotonic dystrophy type 1 microsatellites). The importance of repeat instability in muscular and neurodegenerative disorders (e.g., Huntington disease, Fragile X syndrome, spinocerebellar ataxias, etc.) has recently been the focus of a Special Issue in Nature Reviews Genetics (October 2005, vol. 6, no. 10), highlighting the timeliness and importance of the topic. However, the vast majority of TR loci are found in non-coding regions of the genome that have no known function (‘junk DNA’). Therefore, the functional significance of induced mutation at TRs is uncertain. Furthermore, the mechanisms that result in induced instability at the various types of TR sequences are currently unclear. For some loci it has been demonstrated that induced mutation results from ‘indirect’ effects manifested elsewhere in the genome, and that the consequences of these effects can be stably transmitted to offspring through multiple generations [23,24]. Indirect mutation, and epigenetic transmission of genetic instability to non-exposed descendants, starkly contrast traditional paradigms in genetic toxicology [25]. Lastly, the relationship or correlation between mutation in TR sequences and unique sequence DNA remains a mystery. Although there are many unanswered questions, the application of TR mutation arguably remains the only assay capable of detecting statistically significant increases in germline mutation resulting from exposure to environmental levels of mutagens. Consequently, it
0027-5107/$ – see front matter. Crown Copyright © 2006 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2006.01.006
2
Editorial / Mutation Research 598 (2006) 1–5
stands to reason that a great deal of research emphasis should be placed on: (a) deciphering the mechanisms involved in both spontaneous and induced TR mutation and (b) increasing our understanding of the biological significance of non-functional TR sequence mutation. Furthermore, it is also important to consider that ‘junk DNA’ may have some functional significance (e.g., [26]) and that repetitive DNA may play an important role in the rapid evolution of species [27]. The majority of articles in this special issue of Mutation Research provide an overview of our current understanding of the utility of TR mutation in measuring induced genetic instability, the importance and relevance of TR DNA mutation assays, and the potential mechanisms involved in generating instability across the different repeat types. An introductory commentary by Armour [28] argues the importance of TR DNA, its utility for the study of mutation, and describes the functionality of a subset of TR DNAs. Armour establishes TR regions as effective reporters of genetic instability and induced mutation, and discusses the functional repercussions of these mutations. Gomes-Pereira and Monckton provide a review of instability at microsatellite repeats associated with human genetic disorders [29]. In addition to germline expansion, the authors highlight some exciting new data on chemical modifiers of somatic repeat expansion and suggest that these chemical treatments could potentially be useful as therapeutic tools to modulate disease progression over time. Somers [30] reviews induced expanded simple tandem repeat (ESTR; [31,32]) mutation in the male mouse germline. His review details the discrepancies in the field, including controversy over the timing of induced mutation, and the types and efficiencies of DNA lesions that cause destabilization. The non-targeted nature of induced mutation is discussed in the context of the mouse models that have been used to examine instability. It is clear that the mechanisms that result in ESTR destabilization are unknown and much more work is needed. Barber and Dubrova [33] present data over-viewing radiation-induced genomic instability in the context of trans-generational effects. Work from this pioneering laboratory was the first to show that radiation-induced germline instability can persist through multiple generations. Radiation-induced instability arising in nonexposed progeny poses a fundamental challenge to the way radiation risk assessment is currently undertaken, and suggests that the risks of exposure to ionizing radiation could be greater than previously predicted. It is clear from their review that the molecular and cellular events that contribute to the manifestation of persistent instability are unknown and require further research. Work
from Niwa’s laboratory in the early 1990’s was also ground-breaking in establishing the important potential role of TR in understanding induced genetic instability. Niwa’s review [34] presents further hypotheses to address discrepancies in the timing of induced TR mutation. Niwa argues that mutations arise in the zygote as a result of inheritance of damaged paternal DNA, when DNA is damaged just prior to conception. This hypothesis is supported by his finding of increased mutation at both ESTR loci and the pink-eyed unstable locus of maternal alleles in descendants of irradiated fathers. The delayed effects observed in Niwa’s experiments demonstrate persistent instability, consistent with an epigenetic mechanism, and potentially similar to the mechanisms operating in trans-generational instability. Niwa effectively argues for a possible role of p53 in this response. Clearly more work is needed to decipher the mechanisms operating in both immediate and delayed mutation at TR regions. Cell culture assays facilitate dissecting the mechanistic aspects of repeat mutation. A cell culture assay is used to investigate TR mutation in an original contribution from Polyzos et al. [35]. The authors used a single-molecule PCR approach to measure induced ESTR instability at the Ms6-hm locus after exposure of cultured murine embryonic fibroblast cells to low doses of chemicals with different modes of action. At subtoxic doses of chemicals the authors find that a DNA alkylating agent (N-nitroso-N-ethylurea), a bulky DNA adduct forming agent (benzo(a)pyrene), and a topoisomerase inhibitor (etoposide) caused similar induction of ESTR instability in embryonic fibroblasts. In contrast, a protein kinase inhibitor (okadaic acid) did not cause significant ESTR mutation. The work takes us a step closer to understanding the types of lesions that can result in TR destabilization, and future work will examine a wider spectrum of chemicals. The development of a green fluorescent protein assay using flow cytometry to measure frameshift mutations at TR regions in mouse cell cultures is presented by Healy et al. [36]. The assay not only allows a detailed dose–response investigation of the chemicals that can result in destabilization at repeat units, but also allows the investigation of sequence specificity in the context of instability. Interestingly, treatment with model chemical mutagens or tumour promoters induced statistically significant sequence- and exposuredependent responses. The development of this assay will allow for more detailed mechanistic studies with a broad range of constructs and a variety of chemicals, to provide insight into mechanisms operating in repeat instability. In vitro work described by Wang and Vasquez [37] and Nakagama et al. [38] examines structural conforma-
Editorial / Mutation Research 598 (2006) 1–5
tions that repeat sequences may form that contribute to their instability, and the mechanisms potentially operating in repeat dynamics. Wang and Vasquez provide a review of the role of non-B DNA conformations in repeat mutation. Repetitive DNA elements are more prone to forming such structures, including hairpins, left-handed Z-DNA and intramolecular triplexes, which can lead to both expansion and contraction in allele size. The authors discuss potential mechanisms operating in mutation, including the involvement of mismatch repair proteins, nucleotide excision repair proteins, topoisomerases and structure-specific nucleases. Nakagama et al. [38] examine the molecular mechanisms involved in the maintenance of stability at G-rich repetitive sequences, such as minisatellites and ESTRs. G-rich DNA sequences can adopt a variety of non-canonical DNA structures, including G4 DNA. These sequences can be recombination and mutation hotspots in vivo, and may have the capacity to block the progression of DNA polymerases. The RNA–protein complexes, hnRNP A1 and hnRNP A3, bind to these sequences and can unwind the secondary structures. Therefore, these proteins might facilitate DNA replication at these loci and assist in the maintenance of genomic stability. Reviews by Cederberg and Rannug [39] and by Bichara et al. [40] examine model organisms to dissect the mechanisms operating in TR mutation. Cederberg and Rannug review work examining human minisatellites integrated into the Saccharomyces cerevisiae genome. Both human and yeast minisatellite mutation is driven by meiotic recombination, but the structural changes that are generated by the recombination events differ. In contrast to human minisatellites, inter-allelic transfer of repeats in yeast occurs by conversion, and not crossing over, and several chromatids can be involved in successive recombination events in one meiosis. Intrinsic properties of the minisatellite do not govern instability in meiosis, but do play a role in instability arising in mitosis. The results demonstrate that meiotic mutation in the integrated human minisatellites is highly dependent on genomic location and that a number of genes involved in initiation of recombination and DNA repair are required for mutation induction and maintenance of stability. Bichara et al. [40] review and present new research findings on microsatellite instability in prokaryotes, with emphasis on studies using the model organism Escherichia coli. Both the frequency and types of mutations that occur in E. coli are influenced by the DNA sequence of the repeat, the number of repeat units, and the cellular pathways that process the TR sequences. DNA strand slippage is the general model presented to explain prokaryotic TR instability, with misaligned
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DNA sequences stabilized by favorable base pairing of complementary sequences and by the predisposition of TRs to form relatively stable secondary structures. The work reviews the cellular processes that are potentially involved, including replication, recombination and a variety of DNA repair pathways, and presents new experimental data suggesting that translesion synthesis polymerases (PolII, PolIV and PolV) do not contribute significantly to TR instability in E. coli. The last review in the issue, by Singer et al. [41], examines traditional mouse germ cell mutation assays and their correlation to TR instability and transgenic rodent (TGR) assays. The latter two assays offer considerable advantages over the traditional approaches. An understanding of the correlation among assays, and the relationship between repeat mutation and other types of DNA damage and mutation, is crucial in order to decipher the biological implications of repeat instability. This detailed comparative study examined data in the literature pertaining to the mutagenicity of chemicals and radiation in the dominant lethal assay, the morphological specific locus test, the heritable translocation assay, ESTR and/or TGR mutation assays. Only a handful of mutagens have been examined using several of the methods and the results of the paper demonstrate that development of the TGR and ESTR germline mutation assays for regulatory testing will require more robust and extensive characterization of assay performance. The laboratories represented in this special issue have made important contributions in this field and I am extremely grateful to them for taking the time to contribute. The high quality of the articles is the result of the great care put into the preparation of the manuscripts in addition to the helpful suggestions provided by the referees. I would like to take the opportunity to thank all of the authors and reviewers for what I hope will be a highly regarded review on the status of TR mutation in the context of induced instability. Finally, I would like to express my gratitude to Peter Stambrook and Elsevier for allowing me to act as guest editor. In addition to having been a fantastic learning experience, this has been an invaluable opportunity to bring together opinions and findings from complementary laboratories in what I view as an exciting, dynamic and important research field. References [1] Y.E. Dubrova, A.J. Jeffreys, A.M. Malashenko, Mouse minisatellite mutations induced by ionizing radiation, Nat. Genet. 5 (1993) 92–94. [2] A.J. Jeffreys, V. Wilson, S.L. Thein, Hypervariable ’minisatellite’ regions in human DNA, Nature 314 (1985) 67–73.
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[3] Y.E. Dubrova, M. Plumb, J. Brown, J. Fennelly, P. Bois, D. Goodhead, A.J. Jeffreys, Stage specificity, dose response, and doubling dose for mouse minisatellite germ-line mutation induced by acute radiation, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 6251– 6255. [4] Y.E. Dubrova, V.N. Nesterov, N.G. Krouchinsky, V.A. Ostapenko, R. Neumann, D.L. Neil, A.J. Jeffreys, Human minisatellite mutation rate after the Chernobyl accident, Nature 380 (1996) 683–686. [5] Y.E. Dubrova, V.N. Nesterov, N.G. Krouchinsky, V.A. Ostapenko, G. Vergnaud, F. Giraudeau, J. Buard, A.J. Jeffreys, Further evidence for elevated human minisatellite mutation rate in Belarus eight years after the Chernobyl accident, Mutat. Res. 381 (1997) 267–278. [6] Y.E. Dubrova, R.I. Bersimbaev, L.B. Djansugurova, M.K. Tankimanova, Z. Mamyrbaeva, R. Mustonen, C. Lindholm, M. Hulten, S. Salomaa, Nuclear weapons tests and human germline mutation rate, Science 295 (2002) 1037. [7] Y.E. Dubrova, G. Grant, A.A. Chumak, V.A. Stezhka, A.N. Karakasian, Elevated minisatellite mutation rate in the postchernobyl families from ukraine, Am. J. Hum. Genet. 71 (2002) 801–809. [8] H. Ellegren, G. Lindgren, C.R. Primmer, A.P. Moller, Fitness loss and germline mutations in barn swallows breeding in Chernobyl, Nature 389 (1997) 593–596. [9] O. Kovalchuk, Y.E. Dubrova, A. Arkhipov, B. Hohn, I. Kovalchuk, Wheat mutation rate after Chernobyl, Nature 407 (2000) 583–584. [10] S.H. Rogstad, B. Keane, M.H. Collier, Minisatellite DNA mutation rate in dandelions increases with leaf-tissue concentrations of Cr, Fe, Mn, and Ni, Environ. Toxicol. Chem. 22 (2003) 2093–2099. [11] C.L. Yauk, J.S. Quinn, Multilocus DNA fingerprinting reveals high rate of heritable genetic mutation in herring gulls nesting in an industrialized urban site, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 12137–12141. [12] C.L. Yauk, G.A. Fox, B.E. McCarry, J.S. Quinn, Induced minisatellite germline mutations in herring gulls (Larus argentatus) living near steel mills, Mutat. Res. 452 (2000) 211–218. [13] C.M. Somers, B.E. McCarry, F. Malek, J.S. Quinn, Reduction of particulate air pollution lowers the risk of heritable mutations in mice, Science 304 (2004) 1008–1010. [14] C.M. Somers, C.L. Yauk, P.A. White, C.L. Parfett, J.S. Quinn, Air pollution induces heritable DNA mutations, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 15904–15907. [15] C.A. May, K. Tamaki, R. Neumann, G. Wilson, G. Zagars, A. Pollack, Y.E. Dubrova, A.J. Jeffreys, M.L. Meistrich, Minisatellite mutation frequency in human sperm following radiotherapy, Mutat. Res. 453 (2000) 67–75. [16] J.A. Armour, M.H. Brinkworth, A. Kamischke, Direct analysis by small-pool PCR of MS205 minisatellite mutation rates in sperm after mutagenic therapies, Mutat. Res. 445 (1999) 73–80. [17] A. Kiuru, A. Auvinen, M. Luokkamaki, K. Makkonen, T. Veidebaum, M. Tekkel, M. Rahu, T. Hakulinen, K. Servomaa, T. Rytomaa, R. Mustonen, Hereditary minisatellite mutations among the offspring of Estonian Chernobyl cleanup workers, Radiat. Res. 159 (2003) 651–655. [18] L.A. Livshits, S.G. Malyarchuk, S.A. Kravchenko, G.H. Matsuka, E.M. Lukyanova, Y.G. Antipkin, L.P. Arabskaya, E. Petit, F. Giraudeau, P. Gourmelon, G. Vergnaud, B. Le Guen, Children of chernobyl cleanup workers do not show elevated rates of mutations in minisatellite alleles, Radiat. Res. 155 (2001) 74–80.
[19] C. Satoh, N. Takahashi, J. Asakawa, M. Kodaira, R. Kuick, S.M. Hanash, J.V. Neel, Genetic analysis of children of atomic bomb survivors, Environ. Health Perspect. 104 (Suppl. 3) (1996) 511–519. [20] M. Kodaira, S. Izumi, N. Takahashi, N. Nakamura, No evidence of radiation effect on mutation rates at hypervariable minisatellite loci in the germ cells of atomic bomb survivors, Radiat. Res. 162 (2004) 350–356. [21] M. Stapleton, P.O. Dunn, J. McCarty, A. Secord, L.A. Whittingham, Polychlorinated biphenyl contamination and minisatellite DNA mutation rates of tree swallows, Environ. Toxicol. Chem. 20 (2001) 2263–2267. [22] N. Zheng, D.G. Monckton, G. Wilson, F. Hagemeister, R. Chakraborty, T.H. Connor, M.J. Siciliano, M.L. Meistrich, Frequency of minisatellite repeat number changes at the MS205 locus in human sperm before and after cancer chemotherapy, Environ. Mol. Mutagen. 36 (2000) 134–145. [23] Y.E. Dubrova, M. Plumb, B. Gutierrez, E. Boulton, A.J. Jeffreys, Transgenerational mutation by radiation, Nature 405 (2000) 37. [24] R. Barber, M.A. Plumb, E. Boulton, I. Roux, Y.E. Dubrova, Elevated mutation rates in the germ line of first- and secondgeneration offspring of irradiated male mice, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 6877–6882. [25] B.A. Bridges, Strange goings-on in the mouse germ line, DNA Repair (Amst.) 2 (2003) 1269–1272. [26] P. Andolfatto, Adaptive evolution of non-coding DNA in Drosophila, Nature 437 (2005) 1149–1152. [27] J.W. Fondon III, H.R. Garner, Molecular origins of rapid and continuous morphological evolution, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 18058–18063. [28] J.A.L. Armour, Tandemly repeated DNA: why should anyone care? Mutat Res. 598 (2006) 6–14. [29] M. Gomes-Pereira, D.G. Monckton, Chemical modifiers of unstable expanded simple sequence repeats: what goes up, could come down, Mutat. Res. 598 (2006) 15–34. [30] C.M. Somers, Expanded simple tandem repeat (ESTR) mutation induction in the male germline: lesssons learned from lab mice, Mutat. Res. 598 (2006) 35–49. [31] R. Kelly, M. Gibbs, A. Collick, A.J. Jeffreys, Spontaneous mutation at the hypervariable mouse minisatellite locus Ms6-hm: flanking DNA sequence and analysis of germline and early somatic mutation events, Proc. Biol. Sci. 245 (1991) 235– 245. [32] P. Bois, J. Williamson, J. Brown, Y.E. Dubrova, A.J. Jeffreys, A novel unstable mouse VNTR family expanded from SINE B1 elements, Genomics 49 (1998) 122–128. [33] R.C. Barber, Y.E. Dubrova, The offspring of irradiated parents, are they stable? Mutat Res. 598 (2006) 50–60. [34] O. Niwa, Indirect mechanisms of genomic instability and the biological significance of mutations at tandem repeat loci, Mutat. Res. 598 (2006) 61–72. [35] A. Polyzos, C. Parfett, C. Healy, G.R. Douglas, C.L. Yauk, Instability of expanded simple tandem repeats is induced in cell culture by a variety of agents: N-nitroso-N-ethylurea, benzo(a)pyrene, etoposide and okadaic acid, Mutat. Res. 598 (2006) 73–84. [36] C. Healy, M. Wade, A. McMahon, A. Williams, D. Johnson, C. Parfett, Flow cytometric detection of tandem repeat mutations induced by various chemical classes, Mutat. Res. 598 (2006) 85–102. [37] G. Wang, K.M. Vasquez, Non-B DNA structure-induced genetic instability, Mutat. Res. 598 (2006) 103–119.
Editorial / Mutation Research 598 (2006) 1–5 [38] H. Nakagama, K. Higuchi, E. Tanaka, N. Tsuchiya, K. Nakashima, M. Katahira, H. Fukuda, Molecular mechanisms for maintenance of G-rich short tandem repeats, Mutat. Res. 598 (2006) 120–131. [39] H. Cederberg, U. Rannug, Mechanisms of human minisatellite mutation in yeast, Mutat. Res. 598 (2006) 132–143. [40] M. Bichara, J. Wagner, I.B. Lambert, Mechanisms of Tandem Repeat Instability in Bacteria, Mutat. Res. 598 (2006) 144–163. [41] T.M. Singer, I.B. Lambert, A. Williams, G.R. Douglas, C.L. Yauk, Detection of induced male germline mutation: correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeats, Mutat. Res. 598 (2006) 164–193.
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Carole L. Yauk ∗ Environmental and Occupational Toxicology Division, Health Canada, Environmental Health Centre, Tunney’s Pasture, 0803A, Ottawa, Ont., Canada K1A 0L2 ∗ Tel.:
+1 613 941 7376; fax: +1 613 941 8530. E-mail address: Carole [email protected] Available online 14 February 2006