Abstract
The incorporation of DNA into nucleosomes and higher-order forms of chromatin in vivo creates difficulties with respect to its accessibility for cellular functions such as transcription, replication, repair and recombination. To understand the role of chromatin structure in the process of homologous recombination, we have studied the interaction of nucleoprotein filaments, comprised of RecA protein and ssDNA, with minichromosomes. Using this paradigm, we have addressed how chromatin structure affects the search for homologous DNA sequences, and attempted to distinguish between two mutually exclusive models of DNA-DNA pairing mechanisms. Paradoxically, we found that the search for homologous sequences, as monitored by unwinding of homologous or heterologous duplex DNA, was facilitated by nucleosomes, with no discernible effect on homologous pairing. More importantly, unwinding of minichromosomes required the interaction of nucleoprotein filaments and led to the accumulation of circular duplex DNA sensitive to nuclease Pl. Competition experiments indicated that chromatin templates and naked DNA served as equally efficient targets for homologous pairing. These and other findings suggest that nucleosomes do not impede but rather facilitate the search for homologous sequences and establish, in accordance with one proposed model, that unwinding of duplex DNA precedes alignment of homologous sequences at the level of chromatin. The potential application of this model to investigate the role of chromosomal proteins in the alignment of homologous sequences in the context of cellular recombination is considered.
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References
Adzuma K (1992) Stable synapsis of homologous DNA molecules mediated by E. coli RecA protein involves local exchange of DNA strands. Genes Dev 6:1679–1694
Atcheson E, Esposito RS (1993) Meiotic recombination in yeast. Curr Opinion Genet Dev 3:736–744
Benson FE, Stasiak A, West SC (1994) Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA. EMBO J 13:5764–5771
Bianchi M, DasGupta C, Radding CM (1983) Synapsis and the formation of paranemic joints by E. coli RecA protein. Cell 34:931–939
Bonne-Andrea C, Wong ML, Alberts B (1990) In vitro replication through nucleosomes without histone displacement. Nature 343:719–726
Bramhill D, Kornberg A (1988) Duplex opening by DnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome. Cell 52:743–755
Camerini-Otero RD, Hsieh P (1993) Parallel DNA triplexes, homologous recombination and other homology-dependent interactions. Cell 73:217–233
Cunningham RP, DasGupta C, Shibata T, Radding CM (1980) Homologous pairing in genetic recombination: RecA protein makes joint molecules of gapped and closed circular DNA. Cell 20:223–235
DiCapua C, Muller C (1987) The accessibility to dimethyl sulfate in complexes with RecA protein. EMBO J 6:2493–2498
Dynan WS, Jendrisak JJ, Hager DA, Burgess RR (1981) Purification and characterization of wheat germ DNA topoisomerase I. J Biol Chem 256:5860–5865
Felsenfeld G (1992) Chromatin: an essential part of the transcriptional apparatus. Nature 355:219–224
Flory J, Tsang SS, Muniyappa K (1984) Isolation and visualization of active presynaptic filaments of RecA protein-single stranded DNA. Proc Natl Acad Sci USA 81:7026–7030
Getts RC, Behe MJ (1991) Eukaryotic DNA does not form nucleosomes as readily as some prokaryotic DNA. Nucleic Acids Res 21:5923–5927
Griffith J, Shores GC (1985) RecA protein rapidly crystallizes in the presence of spermidine: a valuable step in its purification and physical characterization. Biochemistry 24:158–162
Germond JE, Hirts B, Oudet P, Gross-Bellard M, Chambon P (1975) Folding of the DNA double helix in chromatin-like structure from Simian Virus 40. Proc Natl Acad Sci USA 72:1843–1847
Jain S, Inman R, Cox MM (1992) Putative three-stranded DNA pairing intermediate in RecA protein-mediated DNA strand exchange. J Biol Chem 267:4215–4222
Jwang B, Radding CM (1992) Torsional stress generated by RecA protein during DNA strand exchange separates strands of a heterologous insert. Proc Natl Acad Sci USA 89:7596–7600
Kleckner N, Padmore R, Bishop DK (1991) Meiotic chromosome metabolism: one view. Cold Spring Harbor Symp Quant Biol 56:729–743
Kotani H, Kmiec E (1994a) Transcription activates RecA-promoted homologous pairing of nucleosomal DNA. Mol Cell Biol 14:1949–1955
Kotani H, Kmiec E (1994b) DNA cruciform facilitate in vitro strand transfer on nucleosomal templates. Mol Gen Genet 243:681–690
Kotani H, Sekiguchi JM, Dutta S, Kmiec E (1994) Genetic recombination of nucleosomal templates is mediated by transcription. Mol Gen Genet 244:410–419
Kowalczykowski S, Eggelston A (1994) Homologous pairing and DNA strand exchange proteins. Annu Rev Biochem 63:991–1043
Krajewski WA, Luchnik AN (1991) Relationship of histone acetylation to DNA topology and transcription. Mol Gen Genet 230:442–448
Krajewski WA, Luchnik AN (1992) High rotational mobility of DNA in animal cells and its modulation by histone acetylation. Mol Gen Genet 231:17–21
Kuhnlein J, Penhoet EE, Linn S (1976) An altered apurinic DNA endonuclease activity in group A and group D xeroderma pigmentosum fibroblasts. Proc Natl Acad Sci USA 73:1169–1173
Kumar AK, Muniyappa K (1992) Use of structure-directed DNA ligands to probe the binding of RecA protein to narrow and wide grooves of DNA and on its ability to promote homologous pairing. J Biol Chem 267:24824–24832
Lichten M, Borts RH, Haber J (1987) Meiotic gene conversion and crossing over between dispersed homologous sequences occurs frequently in Saccharomyces cerevisiae. Genetics 115:233–246
Liu L-F, Wang JC (1987) Supercoiling of the DNA template during transcription. Proc Natl Acad Sci USA 84:7024–7027
Lohman TM, Green JM, Bayer RS (1986) Large scale overproduction and rapid purification of the E. coli ssb gene product: expression of the ssb gene under PL control. Biochemistry 25:21–25
Losa R, Brown DD (1987) A bacteriophage RNA polymerase transcribes in vitro through a nucleosome core without displacing it. Cell 50:801–808
Luchnik AN, Bakayev VV, Zbarsky IB, Georgiev GP (1982) Elastic torsional strain in DNA within a fraction of SV40 minichromosomes: relation to transcriptionally active chromatin. EMBO J 1:1353–1358
Luchnik AK, Bakayev VV, Glaser VM (1983) DNA supercoiling: changes during cellular differentiation and activation of chromatin transcription. Cold Spring Harbor Symp Quant Biol 47:793–801
Luchnik AN, Hisamutadinov TA, Georgiev GP (1989) Inhibition of transcription in eukaryotic cells by X-ray irradiation: relation to the loss of topological constraints in closed DNA loops. Nucleic Acids Res 16:5175–5190
Lutter LC (1989) Thermal unwinding of SV 40 transcription complex. Proc Natl Acad Sci USA 86:8712–8716
McGavin S (1977) A model for the specific pairing of homologous double-stranded nucleic acid molecules during genetic recombination. Heredity 39:15–25
Morse RH, Cantor CR (1985) Nucleosome core particles suppress the thermal untwisting of DNA and adjacent linker DNA. Proc Natl Acad Sci USA 82:4653–4657
Muniyappa K, Williams K, Chase J, Radding CM (1990) Active nucleoprotein filaments of single-stranded binding protein and RecA protein on single-stranded DNA have a regular repeating structure. Nucleic Acids Res 18:3967–3973
Muniyappa K, Ramdas J, Mythili E, Galande S (1991) Homologous pairing between nucleosome cores on a linear duplex DNA and nucleoprotein filaments of RecA protein-single stranded DNA. Biochimie 73:187–190
Ogawa T, Yu X, Shinohara A, Egelman E (1993) Similarity of the yeast Rad51 filament to the bacterial filament. Science 259:1896–1998
Ohta K, Shibata T, Nicolas A (1994) Changes in chromatin structure at recombination initiation sites during yeast meiosis. EMBO J 13:5754–5763
Ohtani T, Shibata T, Iwabuchi M, Watabe H, Iino T, Ando T (1982) ATP-dependent unwinding of double helix in closed circular DNA by RecA protein of E. coli. Nature 299:86–89
O'Neill TE, Roberge M, Bradbury ME (1992) Nucleosome arrays inhibit both initiation and elongation of transcripts by bacteriophage T7 RNA polymerase. J Mol Biol 223:67–78
Paranjape SM, Kamakaka RT, Kadonaga JT (1994) Role of chromatin structure in the regulation of transcription of RNA polymerase II. Anna Rev Biochem 63:265–298
Platz RD, Meistrich ML, Grimes S (1977) Low molecular weight basic proteins in spermatids. Methods Cell Biol 16:297–316
Pryciak P, Varmus HE (1992) Nucleosomes, DNA-binding proteins and DNA sequence modulate retroviral integration target site selection. Cell 69:769–780
Pryciak P, Muller H-P, Varmus HE (1992) SV40 minichromosomes as targets for retroviral integration in vitro. Proc Natl Acad Sci USA 89:9237–9241
Radding CM (1991) Helical interactions in homologous pairing and strand exchange driven by RecA protein. J Biol Chem 266:5355–5358
Ramdas J, Mythili E, Muniyappa K (1989) RecA protein promoted homologous pairing in vitro: pairing between linear duplex DNA bound to HU protein and nucleoprotein filaments of RecA protein-ssDNA. J Biol Chem 254:17395–17400
Ramdas J, Mythili E, Muniyappa K (1991) Nucleosomes on linear duplex DNA allow homologous pairing but prevent strand exchange promoted by RecA protein. Proc Natl Acad Sci USA 88:1344–1348
Roca AI, Cox MM (1990) The RecA protein: structure and function. Crit Rev Biochem Mol Biol 25:415–456
Rould E, Muniyappa K, Radding CM (1992) Unwinding of heterologous DNA by RecA protein during search for homologous sequences. J Mol Biol 226:127–139
Salinas F, Kodadek T (1994) Strand exchange through a DNA-protein complex requires a DNA helicase. Biochem Biophys Res Commun 205:1004–1009
Schutte BC, Cox MM (1988) Homology-dependent underwinding of duplex DNA in RecA protein-generated paranemic complexes. Biochemistry 27:7886–7894
Smerdon MJ, Thoma F (1990) Site-specific DNA repair at the nucleosomal level in a yeast minichromosome. Cell 61:675–684
Stasiak A (1992) Three-stranded DNA structure: is this the secret of homologous recognition? Mol Microbiol 6:3267–3276
Stasiak A, Egelman E (1988) Visualization of recombination reactions. In: Kucherlapati R, Smith GR (eds) Genetic recombination. American Society for Microbiology, Washington DC, pp 265–307
Sung P (1994) Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast Rad51 protein. Science 265:1241–1243
Tsang SS, Muniyappa K, Azhderian A, Gonda D, Radding CM, Flory J, Chase J (1985) Intermediates in homologous pairing promoted by RecA protein. J Mol Biol 185:295–309
Wang Z, Wu X, Friedberg EC (1991) Nucleotide excision repair of DNA by human cell extracts is suppressed in reconstituted nucleosomes. J Biol Chem 266:22472–22478
West S (1992) Enzymes and molecular mechanisms of genetic recombination. Annu Rev Biochem 61:603–640
Wu AM, Bianchi M, DasGupta C, Radding C (1983) Unwinding associated with synapsis of DNA molecules by RecA protein. Proc Natl Acad Sci USA 80:1256–1260
Wu H, Shyy S, Wang JC, Liu L-F (1988) Transcription generates positively and negatively supercoiled domains in the template. Cell 53:433–440
Yu X, Egelman EH (1993) The DNA conformation-induced by the bacteriophage T4 UvsX protein appears identical to the conformation induced by RecA protein. J Mol Biol 232:1–4
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Communicated by G. P. Georgiev
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Ramdas, J., Muniyappa, K. Recognition and alignment of homologous DNA sequences between minichromosomes and single-stranded DNA promoted by RecA protein. Molec. Gen. Genet. 249, 336–348 (1995). https://doi.org/10.1007/BF00290535
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DOI: https://doi.org/10.1007/BF00290535