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DNA excision repair and transcription: implications for genome evolution
DNA excision repair and transcription: implications for genome evolution David T Sullivan Syracuse University, Syracuse, U S A The past two years have seen a substantial increase in knowledge regarding the enzymology of DNA excision repair. These data support a growing body of information which suggests that transcribed nucleotide sequences are preferentially subject to excision repair. It is possible that these mechanisms, or related ones, are relevant to the molecular evolution of sequences that appear not to evolve according to models which do not take into account regional sequence differences in the extent of DNA repair. Current Opinion in Genetics & Development 1995, 5:786-791
Introduction Massive amounts of nucleotide sequence data have been determined during the last two decades, giving great impetus to studies in molecular evolution. Among these is the characterization of evolutionary properties of different sequence classes: synonymous coding nucleotides, non-synonymous coding nucleotides, introns, regions flanking transcription units and pseudogenes. It has become generally accepted that these molecular evolutionary properties are useful in defining a sequence class and its functional state. Sequence divergence can be broken down into two fundamental processes. First, there is structural alteration in DNA followed by a cellular response to that alteration. Second, there are the stochastic and selective forces that will determine whether a new allele will become fixed in a population. Divergence comparisons generally assume, implicitly or explicitly, that repair processes will, on average, be equivalent across sequence classes and therefore not affect the comparisons. Until recently, details regarding DNA repair mechanisms have been have been insufficient to provide compelling reason to doubt these assumptions. However, new results from studies on the biochemistry of one class o f DNA repair system, other data that connect transcription and repair, and the description of some unexpected evolutionary properties of certain sequences, indicate that re-examination of the relationship between mutation, repair and sequence divergence may be in order. These observations and other data suggest that transcribed sequences may be preferentially repaired, thereby raising the possibility that the rules of sequence divergence for transcribed sequences may differ from those which are not transcribed. In this review, I will summarize recent data that connect the machinery for excision repair and basal transcription and describe observations on nucleotide sequence exchanges which at first glance appear atypical. Although a transcription-repair connection might explain these
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examples, as yet there are no direct data connecting transcription-repair and sequence evolution. I offer this review as an example of why the substantial gaps in our present knowledge of nucleic acid metabolism should be considered when drawing evolutionary conclusions.
Common components in repair and transcription machinery Nucleotide excision repair, one of a variety of DNA repair systems, is found in all organisms and can repair a variety of covalent modifications in DNA. Nucleotide excision repair in eukaryotes involves a series of steps: recognition of a structural anomaly in the DNA, endonuclease action 20bp 5' and 4 - 5 b p 3' to the structural anomaly, excision of 27-29 nucleotides (a smaller number in Escherichia agO, D N A synthesis to fill this gap, and finally ligation [1]. Our understanding of the components of the excision repair system has greatly increased in the past two years. Results from three somewhat separate f i e l d s - - D N A repair in yeast, analysis of human genetics diseases and the mechanisms of transcription--have converged to provide an essentially complete description of the components of this system.
DNA repair genes in Saccharomyces cerevisiae Genes involved in DNA repair in S. cerevisiae are referred to as rad genes, as they have nmtant alleles with radiation-sensitive phenotypes. Various of these genes have now been identified, cloned, and their DNA sequences obtained. They fall into three epistasis groups [2]. The RAD3 group is involved in excision repair and includes, in addition to RAD3, RADI, RAD2, RAD4,
RAD7, RADIO, RAD14, RAD16, RAD23, RAD25 and MMS 19.
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DNA excision repair and transcription Sullivan 787 Xeroderma pigmentosum
The second set o f results has come from identification o f the complementing protein components in cell extracts prepared from different individuals having xeroderma pigmentosum or Cockayne's syndrome. Xeroderma pigmentosum is a human disease in which affected individuals have a high incidence of skin cancers and other symptoms. It is inherited as an autosomal recessive condition. The genetics o f this condition have been reviewed recently [3-6] and are summarized briefly here. Progress in characterizing the etiology o f the disease came about when it was recognized that cells from affected individuals were defective in DNA repair and this deficiency could be demonstrated in cell extracts. Furthermore it became evident that several genes are involved, because extracts from different individuals complement in in vitro DNA repair assays. A series o f complementation groups referred to as XP-A through X P - G (and CG-1 and CG-6 for Cockayne's syndrome) were identified by these in vitro tests using human and Chinese hamster ovary cell extracts from radiation-sensitive cell line models. As the genes that encode the proteins o f xeroderma pigmentosum complementation groups were cloned, sequenced and compared to the sequences o f the genes from the yeast RAD3 epistasis group [2-6], it became clear that each group had similar members, thereby establishing the universahty o f a set o f components involved in nucleotide excision repair.
Involvement in DNA repair of components of the basal transcription machinery Although a connection between sequences that are transcribed and those that are preferentially repaired had been demonstrated earlier (see below), the final steps in making the connection between transcription and excision repair began with the finding that components of the basal transcription apparatus are proteins encoded by genes previously identified as being involved in excision repair. Analysis of the multi-component human basal transcription factor 2, also known as TFIIH, which is required for R N A polymerase II transcription, revealed that contains a component with DNA helicase activity. Analysis of the peptide sequence o f the 89 kDa subunit o f TFIIH revealed it to be identical to the human E R C C - 3 gene product (corresponding to the XP-B complementation group) and highly similar to yeast R.AD25 (also known as SSL2) [7]. In addition, the 85 kDa subunit of yeast TFIIH was identified as the product o f the R A D 3 gene and a 50 kDa protein which interacts with TFIIH was found to be the product o f the SSL1 gene, also known to be involved in excision repair [8]. Independently, the RAD25 gene product was shown to be essential for transcription [9]. An essential role for TFIIH in both transcription and excision repair was demonstrated [10°]. These initial studies were followed by numerous genetic and biochemical studies that resulted in characterization o f the components of TFIIH with respect to subunit composition, individual subunit
activities and protein-protein interactions. In general, these studies demonstrated the essential involvement of the products of a set of homologous yeast and human genes in encoding subunits of'-TFIIH or proteins known to interact with TFIIH. These results clearly estabhsh a role for TFIIH in both transcription and excision repair ([11,12°,13,14°, 15,16°, 17,18°,19--21,22°,23°°,24,25 °°, 26°°,27,28]; see annotated references for further information). The molecular details of how TFIIH functions in both repair and transcription are not yet fully understood. Two types o f TFIIH complex have been isolated from yeast and are referred to the TFIIH holoenzyme and the nucleotide excision repairosome. These complexes share some components and have some different, unique components [23°°]. The remaining problem is to understand the relationship between these complexes and how components exchange between the two forms, if they do, and whether one component functions in repair and the other in transcription. Alternatively, a single complex may participate in both processes, shedding some subunits and gaining others as required.
Transcribed sequences are preferentially repaired In addition to genetic and biochemical studies on TFIIH that show common enzymological components in excision repair and transcription, a second set o f results demonstrates that sequences which are transcribed are preferentially subject to excision repair (reviewed in [29°°]). A common assay for repair o f UV-induced cyclopyrimidine dimers is to irradiate cells, extract DNA at successive intervals during recovery, treat the DNA with phage T4 endonuclease V (which digests DNA at cyclopyrimidine dimers), restrict the DNA, and perform a Southern hybridization using an appropriate probe. There is an increase in the relative content o f the relevant restriction fragment as a function of time following irradiation, resulting from its repair, as compared to DNA from nonirradiated cells. This assay has been applied in a number o f experimental contexts that compare excision repair in DNA which is being actively transcribed with D N A which is not being actively transcribed. These include comparison of repair in the dihydrofolate reductase (DHFR) gene to bulk DNA [30], in the D H F R and adenosine deaminase genes to a specific untranscribed genomic sequence [31], in the yeast GAL7 gene under induced or uninduced conditions [32], comparison of repair of the yeast mating type gene 0t at its transcribed position MAT0t and untranscribed position HMLct [33°°], and comparison of excision repair in an actively transcribed gene (DHFR) throughout the cell cycle [34]. In addition, it has been shown that transcription-coupled excision repair, but not all excision repair, is deficient in cells from patients with Cockayne's syndrome [35,36 °] and that
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Genomesand evolution transcription-coupled excision repair does not occur in cells carrying a temperature-sensitive allele of an R N A polymerase gene when studied at the restrictive temperature [32,37]. The conclusion to draw from these results is that a fraction of the excision repair system is specifically committed to and active on the transcribed strand o f sequences that are active. Results from another study designed to examine the relation between transcription and mutation in yeast have recently been published [38"]. In this work, a lys2 frameshift allele was placed under the control o f a GAL promoter and revertants were selected under induction or no induction conditions. Substantially more lys2 revertants were obtained from the induced, transcribed cultures. In part, the increase in mutations was found to be dependent on the presence of a functional R E V 3 gene, a known member of the RAD6 epistasis DNA repair group. These results also seem to support a connection between transcription and repair although the mechanism is not yet clear. One explanation is that many of the mutants derive from an increased frequency of error during repair DNA synthesis of the transcribed region. In this example, transcription correlates with increased sequence change. In another example, summarized below, transcription seems to correlate with decreased sequence change. This apparent paradox might be due to the fact that one example is from yeast and the other is from higher eukaryotes, or that one is seen in mitotically dividing cells and the latter would be a meiotic phenomenon.
Description of unexpected evolutionary properties in specific sequences An issue to address is whether there are aspects o f the molecular mechanisms o f mutation and repair which have an impact on molecular evolutionary comparisons. I offer the recent work connecting excision repair and transcription as one possible example of a mechanism for this. Another way to approach the issue is to ask whether there are cases of sequence evolution which are puzzling and/or unexplained given models that do not involve considerations of mutation or repair. One example appears to be pseudogenes in the genome of Drosophila. In one case, Adh pseudogenes from members o f the repleta species group that are products ofreplicative gene duplication appear to be evolving slowly and show retention of properties similar to their ancestral coding gene [39"]. In another Adh example, retropseudogenes have been found in D. tesserrii and D. yakuba that show retention of reading frame, codon bias and a higher rate o f substitution at synonymous nucleotide positions as compared to non-synonymous positions [40,41°]. This pattern of evolutionary sequence divergence along with comparison o f surrounding sequences has led to the hypothesis that these Adh retropseudogenes may
have become exons in a 'new' functional gene [42]. In both o f the above examples, the existence o f pseudogene transcripts has been reported. Similarly, an Adh retropseudogene has been described in D. subobscura that shows sequence conservation that is higher than expected [43]. My colleagues and I have described a possible retropseudogene ofphosphoglyceromutase in D. melanogaster which remains in frame and is very similar to its functional homologue except for the absence of the first two codons [44]. Evidence from hybridization kinetic analysis shows the Drosophila genome to be composed o f a rapidly diverging and slowly diverging fractions [45-48]. cDNA sequences hybridize principally to DNA of the slowly diverging fraction [49]. Recently, a slower than expected sequence divergence rate has been observed for copia transposable elements in the D. mauritiana and D. simulans genomes [50]. In mammals, it has been reported that synonymous substitution frequencies vary among genes within a species but are similar in homologous genes between species [51"]. One interpretation of these results is that the extent o f transcription and repair which operates on these specific genes is conserved between species but differs for different genes of a given species. Other work has demonstrated that the rate o f silent substitutions varies by at least a factor of three, among a set o f monkey genes [52]. Finally it has been shown that a region of the mouse aprt gene, which is transcribed as the 3' untranslated region o f the mlLNA but which has no known function, evolves at a fourfold slower rate compared to the neighboring downstream untranscribed region due to a reduced rate o f silent mutations [53]. In each o f the above examples, the connection between transcription and repair might serve as the basis for a model to explain what seem to be anomalies of sequence divergence. At this stage, however, a variety of models are possible. I offer the existence of these examples along with the unfolding story on the molecular biology of repair systems to stimulate awareness that this molecular biology may soon be useful in providing a more complete understanding sequence divergence during evolution. One final issue is worth noting. If the association between repair and transcription is relevant to molecular evolutionary considerations, then at least in higher eukaryotes the transcription and repair of concern is that which occurs in the germ line. At present we have only minimal knowledge about germ line transcription. Systems that have been studied in this regard are those in which gametogenesis is accompanied by the formation oflampbrush chromosomes, such as in amphibians. This work has been well sumxnarized in the monograph by Callan [54]. In one specific report it has been conclusively shown that the histone gene transcription unit of amphibian oogenesis is much larger than the one in somatic cells [55]. Therefore, transcript mapping using 1LNA samples derived from somatic cells to identify. regions o f the genome subject to transcription coupled
DNA excision repair and transcription Sullivan 789 repair may be inappropriate for evaluating evolutionary consequences.
repair and in transcription by RNA polymerase II. Nature 1994, 368:769-772. This work reports the purification of human TFIIH. The purified factor was shown to be essential for basal transcription. In addition it complements three different XP cell extracts with regard to in vitro excision repair assays.
Conclusions
11.
The past two years have seen a substantial increase in our understanding o f the enzymology of excision repair. A strong connection between components o f excision repair and transcription is now evident. These results provide a mechanistic basis for other phenomena that also show a connection between the transcription o f a sequence and its extent of repair. A number o f examples of sequences which do not appear to evolve using simple models of sequence evolution are cited. Although it is not yet possible to use the connection between transcription and repair to provide a mechanistic basis for each o f these, explanations may be found in aspects o f the molecular biology of D N A mutation and repair as further details are forthcoming.
12. •
Bardwell AJ, Bardwell L, lyer N, Svejstrup JQ, Feaver W, Kornberg RD, Friedberg EC: Yeast nucleotide excision repair proteins Rad2 and Rad4 interact with RNA polymerase II basal transcription factor b (TFIIH). Mol Cell Biol 1994, 14:3569-3576. This work established, using co-immunoprecipitation assays, that two additional RAD gene products, RAD2 and RAD4 could interact with yeast transcription factor b (TFIIH). 13.
Guzder SN, Sung P, Bailly V, Prakash L, Prakash S: RAD25 is a DNA helicase required for DNA repair and RNA polymerase polymerase II transcription. Nature 1994, 369:578-581. Further analysis of the transcriptional role of RAD25 using the temperature-sensitive allele. The authors demonstrated that this protein has helicase activity. 15.
Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest
16. •
Huang J-C, Svoboda DL, Reardon JT, Sancar A: Human nucleotide excision nuclease removes thymine dimers from DNA by incising the 22nd phosphodiesler bond 5' and the 6 phosphodiester bond 3' to the photodimer. Proc Nat/Acad 5ci USA 1992, 89:3664-3668.
2.
Prakash L, Prakash S, Sung P: DNA repair genes and proteins of Saccharomyces cerevisiae. Annu Rev Genet 1993, 27:33-70.
3.
Reardon JT, Thompson LH, Sancar A: Excision repair in man and the molecular basis of Xeroderma pigmentosom. In DNA and Chromosomes, vol LVIII. Cold Spring Harbor: Cold Spring Harbor Press; 1993:605-618.
4.
Wood RD, Abousseka A, Biggerstaff M, Jones CJ, O'Donovan A, Shivji MKK, Szymkowski DE: Nucleotide excision repair of DNA by mammalian cell extracts and purified proteins. In DNA and Chromosomes, vol LVlII. Cold Spring Harbor; Cold Spring Harbor Press; 1993:625-632.
5.
Hoeijmakers JHJ: Human nucleotide excision repair syndromes: molecular clues to unexpected intricacies. Eur J Cancer 1994, 30A:1912-1921.
6.
Hoeijmakers JHJ, Bootsma D: DNA repair--incisions for excision. 1994, Nature 1994, 371:654-655.
7.
Schaeffer L, Roy R, Humbert S, Moncollin V, Hoeijmakers JHJ, Chambon P, Egly J-M: DNA repair helicase: a component of BTF2(TFIIH) basic transcription factor. Science 1993, 260:58-63.
8.
9.
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Feaver WJ, Svejstrup JQ, Bardwell L, Bardwell AJ, Buratowski S, Gulyas KD, Donahue TF, Friedberg EC, Kornberg RD: Dual roles of a multiprolein complex from S. cerevisiae in transcription and DNA repair. Cell 1993, 75:1379-1387. Qui H, Park E, Prakash L, Prakash S: The Saccharomyces cerevisiae DNA repair gene RAD25 is required for transcription by RNA polymerase II. Genes Dev 1993, 7:2161-2171. Drapkin R, Reardon JT, Ansari A, Huang J-C, Zawel L, Ahn K, Sancar A, Rienberg D: Dual role of TFIIH in DNA excision
Bardwell L, Bardwell AJ, Feaver WJ, Svejstrup JQ, Komberg RD: Yeast RAD3 protein binds directly 1o both SSL2 and SSL1 proteins: Implications for the structure and function of transcription/repair factor b. Proc Natl Acad Sci USA 1994, 91:3926-3930
14. •
References and recommended reading
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Bang DD, Timmermans V, Verhage R, Zeeman AM, Van de Putte P, Brouwer J: Regulation of the Saccharomyces cerevislae DNA repair gene RAD16. Nucleic Acids Res 1995, 23:1679-1685.
Guzder SN, Bailly V, Sung P, Prakash L, Prakash S: Yeast DNA repair protein RAD23 promotes complex formation between transcription factor TFIIH and DNA damage recognition factor RAD14. J Biol Chem 1995, 270:8385-8388.
Humbert S, Van Vuuren H, Lutz Y, Hoeijmakers JHJ, Egly JM, Moncollin V: p44 and p34 subunits of the BFT2/TFIIH transcription factor have homologies with SSL1, a yeast protein involved in repair. £MBO J 1994, 13:2393-2398. This work reports the purification of the p34 and p44 subunits of TFIIH and the generation of monoclonal antibodies against them. The antibodies precipitate the entire human TFIIH complex, inhibit transcription and deplete in vitro excision repair assays. Hence two additional components of TFIIH are shown to also be involved in repair. 17.
Johnson RE, Prakash S, Prakash L: Yeasl DNA repair protein RAD5 that promotes instability of simple repetitive sequences is a DNA-dependent ATPase. J Biol Chem 1994, 269:28259-28262.
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Ma L, Westbroek A, Jochemsen AG, Weeda G, Bosch A, Bootsma D, Hoeijmakers JHJ, Van der Eb AJ: Mutational analysis of ERCC3, which is involved in DNA repair and transcription initiation: identification of domains essential for the DNA repair function. A4ol Cell giol 1994, 14:4126-4134. This work reports the results of in vitro mutagenesis analysis of human ERCC3. The consequences of mutation were assessed by complementation of repair assays and several domains required for this protein to carry out excision repair were identified. 19.
Roy R, Adamczewski JP, Seroz T, Vermeulen W, Tassan JP, Schaeffer L, Nigg EA, Hoeijmakers JHJ, Egly JM: The MO15 cell cycle kinase is associated with the TFIIH transcription DNA repair factor. Cell 1994, 79:1093-1101.
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Schaeffer L, Moncollin V, Roy R, Staub A, Mezzina M, Sarasin A, Weeda G, Hoeijmakers JHJ, EIgy J-M: The ERCC/2 DNA repair protein is associated with the class II BTF2/TFIIH transcription factor. EMBO J 1994, 13:2388-2392.
21.
Shivji MKK, Eker APM, Wood RD: DNA repair defect in xeroderma pigmentosum group C and complementing factor from HeLa cells. J giol Chem ]994, 269:22749-22757.
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Svejstrup JQ, Feaver WJ, Lapointe J, Komberg RD: RNA polymerase transcription factor IIH holoenzyme from yeast. J Biol Chem 1994, 269:28044-28048. This work reports the purification of yeast TFIIH holoenzyme. Nine subunits are found and these are similar to mammalian TFIIH samples.
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Genomes and evolution Svejstrup JQ, Wang ZG, Fearer WJ, Wu XH, Bushnell DA, Donahue TF, Friedberg EC, Kornberg RD: Different forms of TFIIH fur transcription and DNA repair: holo-TFIIH and a nucleotide excision repairosome. Cell 1995, 80:21-28. The latest work from the laboratory heavily involved in the analysis of the biochemistry of the yeast transcription factors. This work reports further studies on purified yeast TFIIH holoenzyme and its separation from a second activity called a repairosome. The holoenzyme was found to consist of a five subunit core, the SSL2 gene product and a complex of three polypeptides that have kinase activity. The repairosome lacks the kinase complex but contains the RAD1, RAD2, RAD4, RAD10 and RAD14 gene products, all of which are known to be involved in repair. The authors lay out the possibility that one complex functions in transcription and the other in repair. This is in opposition to a view that a single complex participates in both processes.
gene throughout the cell cycle in UV irradiated human cells. Mutat Res 1995, 336:181-192.
23. ""
24.
Van Gool AJ, Verhage R, Swagemakers SMA, Van der Putte P, Brouwer J, Troelstra C, Bootsma D, Hoeijmakers JHJ: RAD26, the functional $. cerevisiae homologue of the Cockayne's syndrome B gene. EAdBO J 1994, 13:5361-5369.
25. ••
Van Hoffen A, Venema J, Meschini R, Van Zeeland ASS,, Mullenders LHF: Transcription coupled repair removes both cyclobutane pyrimidine dlmers and 6-4 photoproducts with equal efficiency and in a sequential way from transcribed DNA in Xeroderma pigmentosum group C flbroblasts. EAdBO] 1995, 14:360-367. One of the most recent and detailed papers of the removal of UV-induced lesions in the transcribed strand of DNA using extracts of normal cells and XP-C cells. Transcription-coupled and global repair of UV induced lesions in the adenosine deaminase gene were compared to a specific non-transcribed sequence called 754. 26. ""
Van Vuuren AJ, Vermeulen W, Ma L, Weeda G, Appeldoom E, Jaspers NGJ, Van der Eb AJ, Bootsma D, Hoeijmakers JHJ, Humbert S, Schaeffer L, Egly J-M: Correction of xeroderma pigmentosum repair defects by basal transcription factor BTF2 (TFIIH). EAdBO ] 1994, 13:1645-1653. This work reports the results of injecting purified human TFIIH into repair defective XP-B cells and then recovering repair activity. This demonstrated that the role of TFIIH in repair is direct.
35.
Troelstra C, Van Gool C, de Wit J, Vermeulen W, Bootsma D, Hoeijmakers JHJ: Ercc6, a member of a sub family of putative helicases, is involved in Cockayne's syndrome and preferential repair of active genes. Cell 1992, 71.:939-953.
Kantor GJ, Bastin SA: Repair of some active genes in Cockayne syndrome cells is at the genome overall rate. Murat Res - DNA Repair 1995, 336:223-233. This work establishes that in Cockayne's syndrome the specific aspect of repair that is missing is the transcription-coupled component of excision repair. 36. •
37.
Sweder KS, Hanawalt PC: Preferential repair of cyclobutane dimers in the transcribed strand in yeast chromosomes and plasmids is dependent upon transcription. Proc Nat/Acad 5ci USA 1992, 89:10696-10700.
Datta A, Jinks-Robertson 5: Association of increased spontaneous mutation rates with high levels of transcription in yeast. Science 1995, 268:1616-1618. This work takes a fundamentaJly different approach to assessing the relative roles of transcription and repair. Rather than a biochemically based monitoring of induced lesions, these authors study the consequences of transcription for the generation of revertants of a frame shift mutant and find the reversion frequency is greatly enhanced in sequences that are transcribed. 38. ""
39. "•
Sullivan DT, Starmer WT, Curtiss SW, Menotti-Raymond MA, Yum J: Unusual molecular evolution of an Adh pseudogene in Drosophila. Ado/ Biol Evol 1994, 11:443-458. This work reports an unusual rate and pattern of sequence evolution in the most extensively molecularly and evolutionarily characterised pseudogenes in Drosophila. 40.
Jeffs P, Ashburner M: Processed pseudogenes in Drosophila. Proc Roy Soc Lond B 1991, 244:151-159.
Jeffs PS, Holmes EC, Ashbumer M: The molecular evolution of the alcohol dehydrogenase and alcohol dehydrogenase-related genes in the Drosophila melanogaster species subgroup. Ado/ Biol Evol 1994, 11:287-304. This work presents new data and summarizes the extensive body of knowledge regarding the molecular evolution of Adh in Drosophila including the peculiarities of Adh retropseudogene evolution. 41. •
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Walker LJ, Craig RB, Harris AL, Hickson ID: A role for the human DNA repair enzyme HAP1 in cellular protection against DNA damaging agents and hypoxic stress. Nucleic Acids Res 1994, 22:4884-4889.
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Wang Z, Svejstrup JQ, Feaver WJ, Wu X, Kornberg RD, Friedberg EC: Transcription factor b (TFIIH) is required during nucleotide-excision repair in yeast. Nature 1994, 368:74-75
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Long MY, Langley CH: Natural selection and the origin of jingwei, a chimeric processed functional gene in Drosophila. Science 1993, 260:91-95.
29. Hanawalt PC: DNA repair comes of age. Murat Res-DNA "• Repair 1995, 336:101-113. This is a recent review on many aspects of DNA repair and specifically transcription coupled repair. It is written by the principal contributor to this field and offers an illuminating historical perspective on the variety of repair mechanisms and the interactions in which that they must participate.
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Mellon I, Bohr VA, Smith CA, Hanawalt PC: Preferential DNA repair of an active gene in human cells. Proc Natl Acad 5ci USA 1986, 83:8878-8882.
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Venema J, Mulenders HLF, Natarajan AT, Van Zeeland AA, Mayne LV: The genetic defect in Cockayne's syndrome is associated with a defect in repair of UV-induced DNA damage in transcriptionally active DNA. Proc Nat/Acad 5ci USA 1990, 87:4707-4711.
Powell JR, Caccone A, Gleason JM, Nigro L: Rates of DNA evolution in Drosophila depend on function and developmental stage of expression. Genetics 1993, 133:291-298.
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Werman SD, Davidson EH, Britten RJ: Rapid evolution in a fraction of the Drosophila nuclear genome. J Ado/Evol 1990, 30:281-289.
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Mouchiroud D, Gautier C, Bernardi G: Frequencies of synonymous substitutions in mammals are gene-specific and
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Meniel V, Magana-Schwencke N, Averbeck D: Preferential repair in yeast cells after induction of interstrand cross-links by methoxypsoralen plus UVA. Adutat Res 1995, 329:121-150. This work reports on the transcription coupled repair of UV-psoralen induced cross-links in DNA. The assays use components of the well characterized yeast mating type systems in transcriptionally active and inactive locations. 34.
Lammel L, CarswelI-Crumpton C, Hannawalt PC: Preferential repair of the transcribed strand in the dihydrofolate reductase
DNA excision repair and transcription Sullivan correlated with frequencies of nonsynonymous substitutions. J Mol Evol 1995, 40:107-113. In this study, the extent of synonymous substitutions in a set of 85 homologous genes from rats, mice, cows and humans were compared. Substitution was found to correlate with homologous genes and not the species. In addition a parallel between synonymous and non-synonymous substitutions was also found. The authors raise the possibility thai a connection between repair and transcription levels could provide a basis for these observations. 52.
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D T Sullivan, Syracuse University, Department of Biology, Biological Research Laboratories, 130 College Koad, Syracuse, NY 13244-1270, USA.
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Introduction Massive amounts of nucleotide sequence data have been determined during the last two decades, giving great impetus to studies in molecular evolution. Among these is the characterization of evolutionary properties of different sequence classes: synonymous coding nucleotides, non-synonymous coding nucleotides, introns, regions flanking transcription units and pseudogenes. It has become generally accepted that these molecular evolutionary properties are useful in defining a sequence class and its functional state. Sequence divergence can be broken down into two fundamental processes. First, there is structural alteration in DNA followed by a cellular response to that alteration. Second, there are the stochastic and selective forces that will determine whether a new allele will become fixed in a population. Divergence comparisons generally assume, implicitly or explicitly, that repair processes will, on average, be equivalent across sequence classes and therefore not affect the comparisons. Until recently, details regarding DNA repair mechanisms have been have been insufficient to provide compelling reason to doubt these assumptions. However, new results from studies on the biochemistry of one class o f DNA repair system, other data that connect transcription and repair, and the description of some unexpected evolutionary properties of certain sequences, indicate that re-examination of the relationship between mutation, repair and sequence divergence may be in order. These observations and other data suggest that transcribed sequences may be preferentially repaired, thereby raising the possibility that the rules of sequence divergence for transcribed sequences may differ from those which are not transcribed. In this review, I will summarize recent data that connect the machinery for excision repair and basal transcription and describe observations on nucleotide sequence exchanges which at first glance appear atypical. Although a transcription-repair connection might explain these
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examples, as yet there are no direct data connecting transcription-repair and sequence evolution. I offer this review as an example of why the substantial gaps in our present knowledge of nucleic acid metabolism should be considered when drawing evolutionary conclusions.
Common components in repair and transcription machinery Nucleotide excision repair, one of a variety of DNA repair systems, is found in all organisms and can repair a variety of covalent modifications in DNA. Nucleotide excision repair in eukaryotes involves a series of steps: recognition of a structural anomaly in the DNA, endonuclease action 20bp 5' and 4 - 5 b p 3' to the structural anomaly, excision of 27-29 nucleotides (a smaller number in Escherichia agO, D N A synthesis to fill this gap, and finally ligation [1]. Our understanding of the components of the excision repair system has greatly increased in the past two years. Results from three somewhat separate f i e l d s - - D N A repair in yeast, analysis of human genetics diseases and the mechanisms of transcription--have converged to provide an essentially complete description of the components of this system.
DNA repair genes in Saccharomyces cerevisiae Genes involved in DNA repair in S. cerevisiae are referred to as rad genes, as they have nmtant alleles with radiation-sensitive phenotypes. Various of these genes have now been identified, cloned, and their DNA sequences obtained. They fall into three epistasis groups [2]. The RAD3 group is involved in excision repair and includes, in addition to RAD3, RADI, RAD2, RAD4,
RAD7, RADIO, RAD14, RAD16, RAD23, RAD25 and MMS 19.
© Current Biology Ltd ISSN 0959-437X
DNA excision repair and transcription Sullivan 787 Xeroderma pigmentosum
The second set o f results has come from identification o f the complementing protein components in cell extracts prepared from different individuals having xeroderma pigmentosum or Cockayne's syndrome. Xeroderma pigmentosum is a human disease in which affected individuals have a high incidence of skin cancers and other symptoms. It is inherited as an autosomal recessive condition. The genetics o f this condition have been reviewed recently [3-6] and are summarized briefly here. Progress in characterizing the etiology o f the disease came about when it was recognized that cells from affected individuals were defective in DNA repair and this deficiency could be demonstrated in cell extracts. Furthermore it became evident that several genes are involved, because extracts from different individuals complement in in vitro DNA repair assays. A series o f complementation groups referred to as XP-A through X P - G (and CG-1 and CG-6 for Cockayne's syndrome) were identified by these in vitro tests using human and Chinese hamster ovary cell extracts from radiation-sensitive cell line models. As the genes that encode the proteins o f xeroderma pigmentosum complementation groups were cloned, sequenced and compared to the sequences o f the genes from the yeast RAD3 epistasis group [2-6], it became clear that each group had similar members, thereby establishing the universahty o f a set o f components involved in nucleotide excision repair.
Involvement in DNA repair of components of the basal transcription machinery Although a connection between sequences that are transcribed and those that are preferentially repaired had been demonstrated earlier (see below), the final steps in making the connection between transcription and excision repair began with the finding that components of the basal transcription apparatus are proteins encoded by genes previously identified as being involved in excision repair. Analysis of the multi-component human basal transcription factor 2, also known as TFIIH, which is required for R N A polymerase II transcription, revealed that contains a component with DNA helicase activity. Analysis of the peptide sequence o f the 89 kDa subunit o f TFIIH revealed it to be identical to the human E R C C - 3 gene product (corresponding to the XP-B complementation group) and highly similar to yeast R.AD25 (also known as SSL2) [7]. In addition, the 85 kDa subunit of yeast TFIIH was identified as the product o f the R A D 3 gene and a 50 kDa protein which interacts with TFIIH was found to be the product o f the SSL1 gene, also known to be involved in excision repair [8]. Independently, the RAD25 gene product was shown to be essential for transcription [9]. An essential role for TFIIH in both transcription and excision repair was demonstrated [10°]. These initial studies were followed by numerous genetic and biochemical studies that resulted in characterization o f the components of TFIIH with respect to subunit composition, individual subunit
activities and protein-protein interactions. In general, these studies demonstrated the essential involvement of the products of a set of homologous yeast and human genes in encoding subunits of'-TFIIH or proteins known to interact with TFIIH. These results clearly estabhsh a role for TFIIH in both transcription and excision repair ([11,12°,13,14°, 15,16°, 17,18°,19--21,22°,23°°,24,25 °°, 26°°,27,28]; see annotated references for further information). The molecular details of how TFIIH functions in both repair and transcription are not yet fully understood. Two types o f TFIIH complex have been isolated from yeast and are referred to the TFIIH holoenzyme and the nucleotide excision repairosome. These complexes share some components and have some different, unique components [23°°]. The remaining problem is to understand the relationship between these complexes and how components exchange between the two forms, if they do, and whether one component functions in repair and the other in transcription. Alternatively, a single complex may participate in both processes, shedding some subunits and gaining others as required.
Transcribed sequences are preferentially repaired In addition to genetic and biochemical studies on TFIIH that show common enzymological components in excision repair and transcription, a second set o f results demonstrates that sequences which are transcribed are preferentially subject to excision repair (reviewed in [29°°]). A common assay for repair o f UV-induced cyclopyrimidine dimers is to irradiate cells, extract DNA at successive intervals during recovery, treat the DNA with phage T4 endonuclease V (which digests DNA at cyclopyrimidine dimers), restrict the DNA, and perform a Southern hybridization using an appropriate probe. There is an increase in the relative content o f the relevant restriction fragment as a function of time following irradiation, resulting from its repair, as compared to DNA from nonirradiated cells. This assay has been applied in a number o f experimental contexts that compare excision repair in DNA which is being actively transcribed with D N A which is not being actively transcribed. These include comparison of repair in the dihydrofolate reductase (DHFR) gene to bulk DNA [30], in the D H F R and adenosine deaminase genes to a specific untranscribed genomic sequence [31], in the yeast GAL7 gene under induced or uninduced conditions [32], comparison of repair of the yeast mating type gene 0t at its transcribed position MAT0t and untranscribed position HMLct [33°°], and comparison of excision repair in an actively transcribed gene (DHFR) throughout the cell cycle [34]. In addition, it has been shown that transcription-coupled excision repair, but not all excision repair, is deficient in cells from patients with Cockayne's syndrome [35,36 °] and that
788
Genomesand evolution transcription-coupled excision repair does not occur in cells carrying a temperature-sensitive allele of an R N A polymerase gene when studied at the restrictive temperature [32,37]. The conclusion to draw from these results is that a fraction of the excision repair system is specifically committed to and active on the transcribed strand o f sequences that are active. Results from another study designed to examine the relation between transcription and mutation in yeast have recently been published [38"]. In this work, a lys2 frameshift allele was placed under the control o f a GAL promoter and revertants were selected under induction or no induction conditions. Substantially more lys2 revertants were obtained from the induced, transcribed cultures. In part, the increase in mutations was found to be dependent on the presence of a functional R E V 3 gene, a known member of the RAD6 epistasis DNA repair group. These results also seem to support a connection between transcription and repair although the mechanism is not yet clear. One explanation is that many of the mutants derive from an increased frequency of error during repair DNA synthesis of the transcribed region. In this example, transcription correlates with increased sequence change. In another example, summarized below, transcription seems to correlate with decreased sequence change. This apparent paradox might be due to the fact that one example is from yeast and the other is from higher eukaryotes, or that one is seen in mitotically dividing cells and the latter would be a meiotic phenomenon.
Description of unexpected evolutionary properties in specific sequences An issue to address is whether there are aspects o f the molecular mechanisms o f mutation and repair which have an impact on molecular evolutionary comparisons. I offer the recent work connecting excision repair and transcription as one possible example of a mechanism for this. Another way to approach the issue is to ask whether there are cases of sequence evolution which are puzzling and/or unexplained given models that do not involve considerations of mutation or repair. One example appears to be pseudogenes in the genome of Drosophila. In one case, Adh pseudogenes from members o f the repleta species group that are products ofreplicative gene duplication appear to be evolving slowly and show retention of properties similar to their ancestral coding gene [39"]. In another Adh example, retropseudogenes have been found in D. tesserrii and D. yakuba that show retention of reading frame, codon bias and a higher rate o f substitution at synonymous nucleotide positions as compared to non-synonymous positions [40,41°]. This pattern of evolutionary sequence divergence along with comparison o f surrounding sequences has led to the hypothesis that these Adh retropseudogenes may
have become exons in a 'new' functional gene [42]. In both o f the above examples, the existence o f pseudogene transcripts has been reported. Similarly, an Adh retropseudogene has been described in D. subobscura that shows sequence conservation that is higher than expected [43]. My colleagues and I have described a possible retropseudogene ofphosphoglyceromutase in D. melanogaster which remains in frame and is very similar to its functional homologue except for the absence of the first two codons [44]. Evidence from hybridization kinetic analysis shows the Drosophila genome to be composed o f a rapidly diverging and slowly diverging fractions [45-48]. cDNA sequences hybridize principally to DNA of the slowly diverging fraction [49]. Recently, a slower than expected sequence divergence rate has been observed for copia transposable elements in the D. mauritiana and D. simulans genomes [50]. In mammals, it has been reported that synonymous substitution frequencies vary among genes within a species but are similar in homologous genes between species [51"]. One interpretation of these results is that the extent o f transcription and repair which operates on these specific genes is conserved between species but differs for different genes of a given species. Other work has demonstrated that the rate o f silent substitutions varies by at least a factor of three, among a set o f monkey genes [52]. Finally it has been shown that a region of the mouse aprt gene, which is transcribed as the 3' untranslated region o f the mlLNA but which has no known function, evolves at a fourfold slower rate compared to the neighboring downstream untranscribed region due to a reduced rate o f silent mutations [53]. In each o f the above examples, the connection between transcription and repair might serve as the basis for a model to explain what seem to be anomalies of sequence divergence. At this stage, however, a variety of models are possible. I offer the existence of these examples along with the unfolding story on the molecular biology of repair systems to stimulate awareness that this molecular biology may soon be useful in providing a more complete understanding sequence divergence during evolution. One final issue is worth noting. If the association between repair and transcription is relevant to molecular evolutionary considerations, then at least in higher eukaryotes the transcription and repair of concern is that which occurs in the germ line. At present we have only minimal knowledge about germ line transcription. Systems that have been studied in this regard are those in which gametogenesis is accompanied by the formation oflampbrush chromosomes, such as in amphibians. This work has been well sumxnarized in the monograph by Callan [54]. In one specific report it has been conclusively shown that the histone gene transcription unit of amphibian oogenesis is much larger than the one in somatic cells [55]. Therefore, transcript mapping using 1LNA samples derived from somatic cells to identify. regions o f the genome subject to transcription coupled
DNA excision repair and transcription Sullivan 789 repair may be inappropriate for evaluating evolutionary consequences.
repair and in transcription by RNA polymerase II. Nature 1994, 368:769-772. This work reports the purification of human TFIIH. The purified factor was shown to be essential for basal transcription. In addition it complements three different XP cell extracts with regard to in vitro excision repair assays.
Conclusions
11.
The past two years have seen a substantial increase in our understanding o f the enzymology of excision repair. A strong connection between components o f excision repair and transcription is now evident. These results provide a mechanistic basis for other phenomena that also show a connection between the transcription o f a sequence and its extent of repair. A number o f examples of sequences which do not appear to evolve using simple models of sequence evolution are cited. Although it is not yet possible to use the connection between transcription and repair to provide a mechanistic basis for each o f these, explanations may be found in aspects o f the molecular biology of D N A mutation and repair as further details are forthcoming.
12. •
Bardwell AJ, Bardwell L, lyer N, Svejstrup JQ, Feaver W, Kornberg RD, Friedberg EC: Yeast nucleotide excision repair proteins Rad2 and Rad4 interact with RNA polymerase II basal transcription factor b (TFIIH). Mol Cell Biol 1994, 14:3569-3576. This work established, using co-immunoprecipitation assays, that two additional RAD gene products, RAD2 and RAD4 could interact with yeast transcription factor b (TFIIH). 13.
Guzder SN, Sung P, Bailly V, Prakash L, Prakash S: RAD25 is a DNA helicase required for DNA repair and RNA polymerase polymerase II transcription. Nature 1994, 369:578-581. Further analysis of the transcriptional role of RAD25 using the temperature-sensitive allele. The authors demonstrated that this protein has helicase activity. 15.
Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest
16. •
Huang J-C, Svoboda DL, Reardon JT, Sancar A: Human nucleotide excision nuclease removes thymine dimers from DNA by incising the 22nd phosphodiesler bond 5' and the 6 phosphodiester bond 3' to the photodimer. Proc Nat/Acad 5ci USA 1992, 89:3664-3668.
2.
Prakash L, Prakash S, Sung P: DNA repair genes and proteins of Saccharomyces cerevisiae. Annu Rev Genet 1993, 27:33-70.
3.
Reardon JT, Thompson LH, Sancar A: Excision repair in man and the molecular basis of Xeroderma pigmentosom. In DNA and Chromosomes, vol LVIII. Cold Spring Harbor: Cold Spring Harbor Press; 1993:605-618.
4.
Wood RD, Abousseka A, Biggerstaff M, Jones CJ, O'Donovan A, Shivji MKK, Szymkowski DE: Nucleotide excision repair of DNA by mammalian cell extracts and purified proteins. In DNA and Chromosomes, vol LVlII. Cold Spring Harbor; Cold Spring Harbor Press; 1993:625-632.
5.
Hoeijmakers JHJ: Human nucleotide excision repair syndromes: molecular clues to unexpected intricacies. Eur J Cancer 1994, 30A:1912-1921.
6.
Hoeijmakers JHJ, Bootsma D: DNA repair--incisions for excision. 1994, Nature 1994, 371:654-655.
7.
Schaeffer L, Roy R, Humbert S, Moncollin V, Hoeijmakers JHJ, Chambon P, Egly J-M: DNA repair helicase: a component of BTF2(TFIIH) basic transcription factor. Science 1993, 260:58-63.
8.
9.
10. •
Feaver WJ, Svejstrup JQ, Bardwell L, Bardwell AJ, Buratowski S, Gulyas KD, Donahue TF, Friedberg EC, Kornberg RD: Dual roles of a multiprolein complex from S. cerevisiae in transcription and DNA repair. Cell 1993, 75:1379-1387. Qui H, Park E, Prakash L, Prakash S: The Saccharomyces cerevisiae DNA repair gene RAD25 is required for transcription by RNA polymerase II. Genes Dev 1993, 7:2161-2171. Drapkin R, Reardon JT, Ansari A, Huang J-C, Zawel L, Ahn K, Sancar A, Rienberg D: Dual role of TFIIH in DNA excision
Bardwell L, Bardwell AJ, Feaver WJ, Svejstrup JQ, Komberg RD: Yeast RAD3 protein binds directly 1o both SSL2 and SSL1 proteins: Implications for the structure and function of transcription/repair factor b. Proc Natl Acad Sci USA 1994, 91:3926-3930
14. •
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Van Hoffen A, Venema J, Meschini R, Van Zeeland ASS,, Mullenders LHF: Transcription coupled repair removes both cyclobutane pyrimidine dlmers and 6-4 photoproducts with equal efficiency and in a sequential way from transcribed DNA in Xeroderma pigmentosum group C flbroblasts. EAdBO] 1995, 14:360-367. One of the most recent and detailed papers of the removal of UV-induced lesions in the transcribed strand of DNA using extracts of normal cells and XP-C cells. Transcription-coupled and global repair of UV induced lesions in the adenosine deaminase gene were compared to a specific non-transcribed sequence called 754. 26. ""
Van Vuuren AJ, Vermeulen W, Ma L, Weeda G, Appeldoom E, Jaspers NGJ, Van der Eb AJ, Bootsma D, Hoeijmakers JHJ, Humbert S, Schaeffer L, Egly J-M: Correction of xeroderma pigmentosum repair defects by basal transcription factor BTF2 (TFIIH). EAdBO ] 1994, 13:1645-1653. This work reports the results of injecting purified human TFIIH into repair defective XP-B cells and then recovering repair activity. This demonstrated that the role of TFIIH in repair is direct.
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Kantor GJ, Bastin SA: Repair of some active genes in Cockayne syndrome cells is at the genome overall rate. Murat Res - DNA Repair 1995, 336:223-233. This work establishes that in Cockayne's syndrome the specific aspect of repair that is missing is the transcription-coupled component of excision repair. 36. •
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Datta A, Jinks-Robertson 5: Association of increased spontaneous mutation rates with high levels of transcription in yeast. Science 1995, 268:1616-1618. This work takes a fundamentaJly different approach to assessing the relative roles of transcription and repair. Rather than a biochemically based monitoring of induced lesions, these authors study the consequences of transcription for the generation of revertants of a frame shift mutant and find the reversion frequency is greatly enhanced in sequences that are transcribed. 38. ""
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D T Sullivan, Syracuse University, Department of Biology, Biological Research Laboratories, 130 College Koad, Syracuse, NY 13244-1270, USA.
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