臺灣博碩士論文加值系統

轉錄作用在高度重複性序列及含有高比例GC核苷酸的DNA模板進行時，容易產生RNA:DNA hybrids，即R-loops結構。正常生物體內R-loops並不容易形成，RNase H酵素能針對hybrids上的RNA進行分解，Sen1與Pif1 helicase則能解開RNA:DNA hybrids，或者透過Topoisomerase I改變DNA雙股的纏繞程度，進而降低R-loops形成的可能性；一旦R-loops異常累積時，就可能造成genome instability。先前研究指出，在CAG三核苷酸重複序列亦會產生因轉錄作用所形成的R-loops結構，進一步導致重複序列的不穩定性。本論文主要探討R-loops降低是否會提升重複序列的穩定性，轉錄因子對於R-loops生成與重複序列穩定性的影響，以及不同核苷酸所組成的重複序列是否造成其穩定性的差異。由實驗結果得知，長片段CAG重複序列送入酵母菌中並誘導轉錄進行，DNA重複序列的不穩定性增加，但是此現象在短片段的CAG重複序列DNA則不會發生。進一步研究發現，藉由表現R-loop inhibitors可以降低DNA的不穩定性，因此R-loops確實為造成此DNA不穩定的一個主要因素。然而，我們發現R-loops能在長片段的CAA重複序列產生，但此結構對於CAA穩定性的影響相對較少，這個結果暗示R-loop會誘導DNA不穩定性的發生，但對於不同核苷酸所組成的重複序列會有不同程度的影響。另外，實驗室先前的研究發現在spt4Δ細胞中，RNA polymerase II (RNAP) 無法穩定地通過長片段CAG重複序列，造成RNAP容易從模板上脫落，因此本論文也探討DNA穩定性是否受到轉錄因子的影響。初步的實驗結果顯示spt4Δ與wild-type在R-loops數量上並沒有明顯差異，但spt4Δ的穩定性卻比WT好，代表R-loops雖然是造成不穩定性的要素，但轉錄因子也會決定不穩定性的程度。目前研究結果顯示，R-loops確實會造成三核苷酸重複序列的不穩定性，並且重複序列的組成以及轉錄因子也會影響到R-loops所誘導的不穩定性。

RNA:DNA hybrids, known as R-loops, are peculiar nucleic acid structures that are generated under transcriptional process; in particular, for these genes with highly repetitive or GC rich sequences. R-loops can be dynamically resolved by a variety of cellular proteins and the persistence of RNA:DNA hybrids has been proposed as a negative factor to influence genome integrity. Recently, R-loops were detected on CAG trinucleotide repeats (TNR) and contributed DNA instability as a consequence of contraction of repetitive sequences. Here, using yeast cells as a model system, we analyzed the integrity of repetitive sequences and found CAG repeat instability is promoted by transcription per se and is dependent on the length of repetitive sequence. This DNA instability is ameliorated by ectopic expression of multiple proteins that bear the enzymatic activity to resolve R-loops. Additionally, we discovered the extent of DNA instability is influenced by the composition of nucleotide sequences. Furthermore, we found a differential effect of transcription elongation factors on the DNA instability of CAG repeats. Our collected results demonstrated the causal effect of R-loops on TNR instability, and also revealed the contribution of transcription elongation factors and TNR nucleotide sequences to this transcription-induced DNA instability.

摘要 i
Abstract ii
英文縮寫表 vii
前言 1
三核苷酸重複序列與人類疾病 1
轉錄作用與重複序列的不穩定性 2
R-loops結構與生成機制 3
R-loop在細胞中的功能 4
R-loops誘導重複序列的不穩定性 (repeat instability) 5
細胞中的R-loop inhibitors 5
實驗目的 8
材料與方法 9
材料 9
一、 質體 9
二、 酵母菌菌株 11
三、 核酸引子 13
四、 培養液與培養基 15
五、 抗體 17
方法 17
一、 大腸桿菌的質體轉型與抽取 17
二、 穩定性分析 17
三、 酵母菌的質體轉型 (LiOAc/DMSO method) 18
四、 酵母菌DNA之抽取 (Glass beads method) 19
五、 南方墨點法 (Southern blot) 20
六、 酵母菌蛋白質之萃取 21
七、 SDS-PAGE電泳與西方墨點法 (western blot) 22
八、 酵母菌基因剔除株之建立 22
九、 R-loop indicator建構 (GFP-RNH1-D193N) 23
十、 染色質免疫沉澱 (Chromatin immunoprecipitation, ChIP) 24
十一、 酵母菌點菌分析 (Spot assay) 25
結果 27
討論 33
參考文獻 37
圖表
Figure 1. 46
Figure 2. 48
Figure 3. 50
Figure 4. 52
Figure 5. 54
Figure 6. 55
Figure 7. 57
Figure S1. 58
Figure S2.. 59
Figure S3.. 60
Table 1. 61

Aguilera, A., and Garcia-Muse, T. (2012). R loops: from transcription byproducts to threats to genome stability. Mol Cell 46, 115-124.

Aguilera, A., and Gomez-Gonzalez, B. (2008). Genome instability: a mechanistic view of its causes and consequences. Nat Rev Genet 9, 204-217.

Allender, C.J., Allainguillaume, J., Lynn, J., and King, G.J. (2007). Simple sequence repeats reveal uneven distribution of genetic diversity in chloroplast genomes of Brassica oleracea L. and (n = 9) wild relatives. Theor Appl Genet 114, 609-618.

Bochman, M.L., Sabouri, N., and Zakian, V.A. (2010). Unwinding the functions of the Pif1 family helicases. DNA Repair (Amst) 9, 237-249.

Boule, J.B., and Zakian, V.A. (2007). The yeast Pif1p DNA helicase preferentially unwinds RNA DNA substrates. Nucleic Acids Res 35, 5809-5818.

Bowater, R.P., Jaworski, A., Larson, J.E., Parniewski, P., and Wells, R.D. (1997). Transcription increases the deletion frequency of long CTG.CAG triplet repeats from plasmids in Escherichia coli. Nucleic Acids Res 25, 2861-2868.

Brook, J.D., McCurrach, M.E., Harley, H.G., Buckler, A.J., Church, D., Aburatani, H., Hunter, K., Stanton, V.P., Thirion, J.P., Hudson, T., et al. (1992). Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member. Cell 68, 799-808.

Budd, M.E., Reis, C.C., Smith, S., Myung, K., and Campbell, J.L. (2006). Evidence suggesting that Pif1 helicase functions in DNA replication with the Dna2 helicase/nuclease and DNA polymerase delta. Mol Cell Biol 26, 2490-2500.

Campuzano, V., Montermini, L., Molto, M.D., Pianese, L., Cossee, M., Cavalcanti, F., Monros, E., Rodius, F., Duclos, F., Monticelli, A., et al. (1996). Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion. Science 271, 1423-1427.

Cerritelli, S.M., and Crouch, R.J. (2009). Ribonuclease H: the enzymes in eukaryotes. FEBS J 276, 1494-1505.

Champoux, J.J. (2001). DNA topoisomerases: structure, function, and mechanism. Annu Rev Biochem 70, 369-413.

Chavez, S., Beilharz, T., Rondon, A.G., Erdjument-Bromage, H., Tempst, P., Svejstrup, J.Q., Lithgow, T., and Aguilera, A. (2000). A protein complex containing Tho2, Hpr1, Mft1 and a novel protein, Thp2, connects transcription elongation with mitotic recombination in Saccharomyces cerevisiae. EMBO J 19, 5824-5834.

Chen, Y.Z., Hashemi, S.H., Anderson, S.K., Huang, Y., Moreira, M.C., Lynch, D.R., Glass, I.A., Chance, P.F., and Bennett, C.L. (2006). Senataxin, the yeast Sen1p orthologue: characterization of a unique protein in which recessive mutations cause ataxia and dominant mutations cause motor neuron disease. Neurobiol Dis 23, 97-108.

Cleary, J.D., Nichol, K., Wang, Y.H., and Pearson, C.E. (2002). Evidence of cis-acting factors in replication-mediated trinucleotide repeat instability in primate cells. Nat Genet 31, 37-46.

Dion, V. (2014). Tissue specificity in DNA repair: lessons from trinucleotide repeat instability. Trends Genet 30, 220-229.

Drolet, M. (2006). Growth inhibition mediated by excess negative supercoiling: the interplay between transcription elongation, R-loop formation and DNA topology. Mol Microbiol 59, 723-730.

Drolet, M., Phoenix, P., Menzel, R., Masse, E., Liu, L.F., and Crouch, R.J. (1995). Overexpression of RNase H partially complements the growth defect of an Escherichia coli delta topA mutant: R-loop formation is a major problem in the absence of DNA topoisomerase I. Proc Natl Acad Sci U S A 92, 3526-3530.

El Hage, A., French, S.L., Beyer, A.L., and Tollervey, D. (2010). Loss of Topoisomerase I leads to R-loop-mediated transcriptional blocks during ribosomal RNA synthesis. Genes Dev 24, 1546-1558.

Fresco, J.R., and Alberts, B.M. (1960). The Accommodation of Noncomplementary Bases in Helical Polyribonucleotides and Deoxyribonucleic Acids. Proc Natl Acad Sci U S A 46, 311-321.

Ginno, P.A., Lott, P.L., Christensen, H.C., Korf, I., and Chedin, F. (2012). R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters. Mol Cell 45, 814-825.

Grabczyk, E., Mancuso, M., and Sammarco, M.C. (2007). A persistent RNA.DNA hybrid formed by transcription of the Friedreich ataxia triplet repeat in live bacteria, and by T7 RNAP in vitro. Nucleic Acids Res 35, 5351-5359.

Guldener, U., Heck, S., Fielder, T., Beinhauer, J., and Hegemann, J.H. (1996). A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24, 2519-2524.

Huang, F.T., Yu, K., Hsieh, C.L., and Lieber, M.R. (2006). Downstream boundary of chromosomal R-loops at murine switch regions: implications for the mechanism of class switch recombination. Proc Natl Acad Sci U S A 103, 5030-5035.

Huertas, P., and Aguilera, A. (2003). Cotranscriptionally formed DNA:RNA hybrids mediate transcription elongation impairment and transcription-associated recombination. Mol Cell 12, 711-721.

Itoh, T., and Tomizawa, J. (1980). Formation of an RNA primer for initiation of
replication of ColE1 DNA by ribonuclease H. Proc Natl Acad Sci U S A 77, 2450-2454.

Jasinska, A., and Krzyzosiak, W.J. (2004). Repetitive sequences that shape the human transcriptome. FEBS Lett 567, 136-141.

Jung, J., and Bonini, N. (2007). CREB-binding protein modulates repeat instability in a Drosophila model for polyQ disease. Science 315, 1857-1859.

Kashi, Y., and King, D.G. (2006). Simple sequence repeats as advantageous mutators in evolution. Trends Genet 22, 253-259.

Klein, B.J., Bose, D., Baker, K.J., Yusoff, Z.M., Zhang, X., and Murakami, K.S. (2011). RNA polymerase and transcription elongation factor Spt4/5 complex structure. Proc Natl Acad Sci U S A 108, 546-550.

Kremer, E.J., Pritchard, M., Lynch, M., Yu, S., Holman, K., Baker, E., Warren, S.T., Schlessinger, D., Sutherland, G.R., and Richards, R.I. (1991). Mapping of DNA instability at the fragile X to a trinucleotide repeat sequence p(CCG)n. Science 252, 1711-1714.

La Spada, A.R., and Taylor, J.P. (2010). Repeat expansion disease: progress and puzzles in disease pathogenesis. Nat Rev Genet 11, 247-258.

Lahaye, A., Stahl, H., Thines-Sempoux, D., and Foury, F. (1991). PIF1: a DNA helicase in yeast mitochondria. Embo j 10, 997-1007.

Lazzaro, F., Novarina, D., Amara, F., Watt, D.L., Stone, J.E., Costanzo, V., Burgers, P.M., Kunkel, T.A., Plevani, P., and Muzi-Falconi, M. (2012). RNase H and postreplication repair protect cells from ribonucleotides incorporated in DNA. Mol Cell 45, 99-110.

Li, X., and Manley, J.L. (2005). New talents for an old acquaintance: the SR protein splicing factor ASF/SF2 functions in the maintenance of genome stability. Cell Cycle 4, 1706-1708.

Li, X., and Manley, J.L. (2006). Cotranscriptional processes and their influence on genome stability. Genes Dev 20, 1838-1847.

Lin, Y., Dent, S.Y., Wilson, J.H., Wells, R.D., and Napierala, M. (2010). R loops stimulate genetic instability of CTG.CAG repeats. Proc Natl Acad Sci U S A 107, 692-697.

Lin, Y., Dion, V., and Wilson, J.H. (2006). Transcription promotes contraction of CAG repeat tracts in human cells. Nat Struct Mol Biol 13, 179-180.

Lin, Y., Hubert, L., Jr., and Wilson, J.H. (2009). Transcription destabilizes triplet repeats. Mol Carcinog 48, 350-361.

Lin, Y., and Wilson, J.H. (2007). Transcription-induced CAG repeat contraction in human cells is mediated in part by transcription-coupled nucleotide excision repair. Mol Cell Biol 27, 6209-6217.

Liquori, C.L., Ricker, K., Moseley, M.L., Jacobsen, J.F., Kress, W., Naylor, S.L., Day, J.W., and Ranum, L.P. (2001). Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9. Science 293, 864-867.

Liu, C.R., Chang, C.R., Chern, Y., Wang, T.H., Hsieh, W.C., Shen, W.C., Chang, C.Y., Chu, I.C., Deng, N., Cohen, S.N., et al. (2012). Spt4 is selectively required for transcription of extended trinucleotide repeats. Cell 148, 690-701.

Liu, L.F., and Wang, J.C. (1987). Supercoiling of the DNA template during transcription. Proc Natl Acad Sci U S A 84, 7024-7027.

Lopez Castel, A., Cleary, J.D., and Pearson, C.E. (2010). Repeat instability as the basis for human diseases and as a potential target for therapy. Nat Rev Mol Cell Biol 11, 165-170.

Mahadevan, M., Tsilfidis, C., Sabourin, L., Shutler, G., Amemiya, C., Jansen, G., Neville, C., Narang, M., Barcelo, J., O'Hoy, K., et al. (1992). Myotonic dystrophy mutation: an unstable CTG repeat in the 3' untranslated region of the gene. Science 255, 1253-1255.

Mangiarini, L., Sathasivam, K., Mahal, A., Mott, R., Seller, M., and Bates, G.P. (1997). Instability of highly expanded CAG repeats in mice transgenic for the Huntington's disease mutation. Nat Genet 15, 197-200.

Martinez-Rucobo, F.W., Sainsbury, S., Cheung, A.C., and Cramer, P. (2011). Architecture of the RNA polymerase-Spt4/5 complex and basis of universal transcription processivity. EMBO J 30, 1302-1310.

Matsuura, T., Yamagata, T., Burgess, D.L., Rasmussen, A., Grewal, R.P., Watase, K., Khajavi, M., McCall, A.E., Davis, C.F., Zu, L., et al. (2000). Large expansion of the ATTCT pentanucleotide repeat in spinocerebellar ataxia type 10. Nat Genet 26, 191-194.

McMurray, C.T. (2010). Mechanisms of trinucleotide repeat instability during human development. Nat Rev Genet 11, 786-799.

Mirkin, S.M. (2007). Expandable DNA repeats and human disease. Nature 447, 932-940.

Mischo, H.E., Gomez-Gonzalez, B., Grzechnik, P., Rondon, A.G., Wei, W., Steinmetz, L., Aguilera, A., and Proudfoot, N.J. (2011). Yeast Sen1 helicase protects the genome from transcription-associated instability. Mol Cell 41, 21-32.
Mizuta, R., Mizuta, M., and Kitamura, D. (2005). Guanine is indispensable for immunoglobulin switch region RNA-DNA hybrid formation. J Electron Microsc (Tokyo) 54, 403-408.

Moreira, M.C., Klur, S., Watanabe, M., Nemeth, A.H., Le Ber, I., Moniz, J.C., Tranchant, C., Aubourg, P., Tazir, M., Schols, L., et al. (2004). Senataxin, the ortholog of a yeast RNA helicase, is mutant in ataxia-ocular apraxia 2. Nat Genet 36, 225-227.

Owen, B.A., Yang, Z., Lai, M., Gajec, M., Badger, J.D., 2nd, Hayes, J.J., Edelmann, W., Kucherlapati, R., Wilson, T.M., and McMurray, C.T. (2005). (CAG)(n)-hairpin DNA binds to Msh2-Msh3 and changes properties of mismatch recognition. Nat Struct Mol Biol 12, 663-670.

Pearson, C.E., Ewel, A., Acharya, S., Fishel, R.A., and Sinden, R.R. (1997). Human MSH2 binds to trinucleotide repeat DNA structures associated with neurodegenerative diseases. Hum Mol Genet 6, 1117-1123.

Pearson, C.E., Nichol Edamura, K., and Cleary, J.D. (2005). Repeat instability: mechanisms of dynamic mutations. Nat Rev Genet 6, 729-742.

Pohjoismaki, J.L., Holmes, J.B., Wood, S.R., Yang, M.Y., Yasukawa, T., Reyes, A., Bailey, L.J., Cluett, T.J., Goffart, S., Willcox, S., et al. (2010). Mammalian mitochondrial DNA replication intermediates are essentially duplex but contain extensive tracts of RNA/DNA hybrid. J Mol Biol 397, 1144-1155.

Polak, U., McIvor, E., Dent, S.Y., Wells, R.D., and Napierala, M. (2013). Expanded complexity of unstable repeat diseases. Biofactors 39, 164-175.

Prado, F., and Aguilera, A. (2005). Impairment of replication fork progression mediates RNA polII transcription-associated recombination. EMBO J 24, 1267-1276.

Reddy, K., Tam, M., Bowater, R.P., Barber, M., Tomlinson, M., Nichol Edamura, K., Wang, Y.H., and Pearson, C.E. (2011). Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats. Nucleic Acids Res 39, 1749-1762.

Richard, G.F., Kerrest, A., and Dujon, B. (2008). Comparative genomics and molecular dynamics of DNA repeats in eukaryotes. Microbiol Mol Biol Rev 72, 686-727.
Roberts, R.W., and Crothers, D.M. (1992). Stability and properties of double and triple helices: dramatic effects of RNA or DNA backbone composition. Science 258, 1463-1466.

Ross, C.A. (2002). Polyglutamine pathogenesis: emergence of unifying mechanisms for Huntington's disease and related disorders. Neuron 35, 819-822.

Roy, D., and Lieber, M.R. (2009). G clustering is important for the initiation of transcription-induced R-loops in vitro, whereas high G density without clustering is sufficient thereafter. Mol Cell Biol 29, 3124-3133.

Schulz, V.P., and Zakian, V.A. (1994). The saccharomyces PIF1 DNA helicase inhibits telomere elongation and de novo telomere formation. Cell 76, 145-155.

Schweitzer, J.K., and Livingston, D.M. (1999). The effect of DNA replication mutations on CAG tract stability in yeast. Genetics 152, 953-963.

Shaw, N.N., and Arya, D.P. (2008). Recognition of the unique structure of DNA:RNA hybrids. Biochimie 90, 1026-1039.

Skourti-Stathaki, K., Proudfoot, N.J., and Gromak, N. (2011). Human senataxin resolves RNA/DNA hybrids formed at transcriptional pause sites to promote Xrn2-dependent termination. Mol Cell 42, 794-805.

Steinmetz, E.J., Conrad, N.K., Brow, D.A., and Corden, J.L. (2001). RNA-binding protein Nrd1 directs poly(A)-independent 3'-end formation of RNA polymerase II transcripts. Nature 413, 327-331.

Strasser, K., Masuda, S., Mason, P., Pfannstiel, J., Oppizzi, M., Rodriguez-Navarro, S., Rondon, A.G., Aguilera, A., Struhl, K., Reed, R., et al. (2002). TREX is a conserved complex coupling transcription with messenger RNA export. Nature 417, 304-308.

Streisinger, G., Okada, Y., Emrich, J., Newton, J., Tsugita, A., Terzaghi, E., and Inouye, M. (1966). Frameshift mutations and the genetic code. This paper is dedicated to Professor Theodosius Dobzhansky on the occasion of his 66th birthday. Cold Spring Harb Symp Quant Biol 31, 77-84.

Sugimoto, N., Nakano, S., Katoh, M., Matsumura, A., Nakamuta, H., Ohmichi, T., Yoneyama, M., and Sasaki, M. (1995). Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes. Biochemistry 34, 11211-11216.

Suraweera, A., Lim, Y., Woods, R., Birrell, G.W., Nasim, T., Becherel, O.J., and Lavin, M.F. (2009). Functional role for senataxin, defective in ataxia oculomotor apraxia type 2, in transcriptional regulation. Hum Mol Genet 18, 3384-3396.

Toth, G., Gaspari, Z., and Jurka, J. (2000). Microsatellites in different eukaryotic genomes: survey and analysis. Genome Res 10, 967-981.

Tracy, R.B., Hsieh, C.L., and Lieber, M.R. (2000). Stable RNA/DNA hybrids in the mammalian genome: inducible intermediates in immunoglobulin class switch recombination. Science 288, 1058-1061.

Tuduri, S., Crabbe, L., Conti, C., Tourriere, H., Holtgreve-Grez, H., Jauch, A., Pantesco, V., De Vos, J., Thomas, A., Theillet, C., et al. (2009). Topoisomerase I suppresses genomic instability by preventing interference between replication and transcription. Nat Cell Biol 11, 1315-1324.

Ursic, D., Himmel, K.L., Gurley, K.A., Webb, F., and Culbertson, M.R. (1997). The yeast SEN1 gene is required for the processing of diverse RNA classes. Nucleic Acids Res 25, 4778-4785.

Verkerk, A.J., Pieretti, M., Sutcliffe, J.S., Fu, Y.H., Kuhl, D.P., Pizzuti, A., Reiner, O., Richards, S., Victoria, M.F., Zhang, F.P., et al. (1991). Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell 65, 905-914.

Wang, G., and Vasquez, K.M. (2009). Models for chromosomal replication-independent non-B DNA structure-induced genetic instability. Mol Carcinog 48, 286-298.

Wells, R.D., Parniewski, P., Pluciennik, A., Bacolla, A., Gellibolian, R., and Jaworski, A. (1998). Small slipped register genetic instabilities in Escherichia coli in triplet repeat sequences associated with hereditary neurological diseases. J Biol Chem 273, 19532-19541.

Westover, K.D., Bushnell, D.A., and Kornberg, R.D. (2004). Structural basis of transcription: nucleotide selection by rotation in the RNA polymerase II active center. Cell 119, 481-489.

Wierdl, M., Greene, C.N., Datta, A., Jinks-Robertson, S., and Petes, T.D. (1996). Destabilization of simple repetitive DNA sequences by transcription in yeast. Genetics 143, 713-721.

Wu, H., Lima, W.F., and Crooke, S.T. (2001). Investigating the structure of human RNase H1 by site-directed mutagenesis. J Biol Chem 276, 23547-23553.

Xu, B., and Clayton, D.A. (1995). A persistent RNA-DNA hybrid is formed during transcription at a phylogenetically conserved mitochondrial DNA sequence. Mol Cell Biol 15, 580-589.

Yu, K., Chedin, F., Hsieh, C.L., Wilson, T.E., and Lieber, M.R. (2003). R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells. Nat Immunol 4, 442-451.

Yu, K., Roy, D., Huang, F.T., and Lieber, M.R. (2006). Detection and structural analysis of R-loops. Methods Enzymol 409, 316-329.

Zhou, J.Q. (2000). Pif1p Helicase, a Catalytic Inhibitor of Telomerase in Yeast. Science 289, 771-774.

Zoghbi, H.Y., and Orr, H.T. (2000). Glutamine repeats and neurodegeneration. Annu Rev Neurosci 23, 217-247.