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International Journal of Radiation Biology Volume 77, 2001 - Issue 5
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Research Article

RPA facilitates rejoining of DNA double-strand breaks in an in vitro assay utilizing genomic DNA as substrate

R. Perrault Division of Experimental Radiation Oncology, Department of Radiation Oncology, Kimmel Cancer Center, Jefferson Medical College, Philadelphia, PA 19107, USA
,
Nge Cheong Division of Experimental Radiation Oncology, Department of Radiation Oncology, Kimmel Cancer Center, Jefferson Medical College, Philadelphia, PA 19107, USA
,
Huichen Wang Division of Experimental Radiation Oncology, Department of Radiation Oncology, Kimmel Cancer Center, Jefferson Medical College, Philadelphia, PA 19107, USA
,
Hongyan Wang Division of Experimental Radiation Oncology, Department of Radiation Oncology, Kimmel Cancer Center, Jefferson Medical College, Philadelphia, PA 19107, USA
&
G. Iliakis Department of Radiation Oncology, Jefferson Medical College, Thompson Building Rm B-1, Philadelphia, PA 19107, USA
Pages 593-607 | Published online: 03 Jul 2009

  • extract (NE) and SalI digested plasmid DNA as a sub
  • strate. Reactions were incubated at 25ßC for 1 h. (A) Asaad, N. A., Zeng, Z.-C., Guan, J., Thacker, J. and Iliakis,
  • EÚect of increasing amounts of recombinant RPA in G., 2000, Homologous recombination as a potential target
  • reactions assembled with 10 mg NE. (B) EÚect of 10 mg forcaÚeineradiosensitization inmammaliancells:
  • RPA in reactions assembled with diÚerent amounts of reduced caÚeine radiosensitization in XRCC2 and
  • NE as indicated. It is evident that RPA has no eÚect on XRCC3 mutants. Oncogene, 19, 5788-5800.
  • DNA end-joining in this assay.
  • Badie, C., Iliakis, G., Foray, N., Alsbeih, G., Cedervall, B., Chavaudra, N., Pantelias, G., Arlett, C. and Malaise, E. P., 1995, Induction and rejoining of DNA
  • to achieve similar levels of processing (Loebrich et al. double-strand breaks and interphase chromosome breaks after exposure to X rays in one normal and two hypersen-
  • 1995, Cheong and Iliakis 1997). sitivie human ? broblast cell lines. Radiation Research, 144,It is notable that a role for RPA in DSB rejoining 26-35.
  • could only be established with assays utilizing irradi-Bases, R., Hamori, I., Piazza, L., Maio, J. and Mendez, F.,
  • ated genomic DNA as a substrate. RPA did not have 1990, DNA base and strand damage in X-irradiated
  • a measurable role in the rejoining of restriction monkey CV-1 cells: in? uence of pretreatment using small doses of radiation. International Journal of Radiation Biology,
  • endonuclease induced breaks in plasmid DNA. This 58, 35-54.
  • result is in line with earlier observations suggesting Baumann, P. and West, S. C., 1997, The human Rad51 protein:
  • diÚerent enzymatic requirements for DSB rejoining polarity of strand transfer and stimulation by hRP-A.
  • in plasmid-based and genomic DNA based assays EMBO Journal, 16, 5198- 5206.
  • (Cheong et al. 1998), and emphasizes the potential
  • Baumann, P. and West, S. C., 1998a, DNA end-joining catalysed by human cell-free extracts. Proceedings of the Nationaldiversity of the rejoining process (Johnson and Academy of Sciences, USA, 95, 14 066-14 070.Fairman 1996, Mason et al. 1996). It also points to
  • Baumann, P. and West, S. C., 1998b, Role of the humanthe usefulness of the genomic DNA assay in the RAD51 protein in homologous recombination and
  • elucidation of processes of DSB rejoining likely to be double-stranded-break repair. Trends in Biochemical Sciences,
  • operational in vivo. Although, the genomic DNA assay 23, 247-251. Boe, S.-O., Sodroski, J., Helland, D. E. and Farnet, C. M.,
  • operates optimally when assembled with cytoplasmic
  • 1995, DNA end-joining in extracts from human cells.induced DNA double-strand breaks in human cell nuclei. International Journal of Radiation Biology, 68, 447-457.
  • Biochemical and Biophysical Research Communications, 215,
  • 987-993.Gasior, S. L., Wong, A. K., Kora, Y., Shinohara, A. and Bishop, D. K., 1998, Rad52 associates with RPA andCheong, N. and Iliakis, G., 1997, In vitro rejoining of double
  • strand breaks induced in cellular DNA by bleomycin andFunctions with Rad55 and Rad57 to assemble meiotic recombination complexes. Genes and Development, 12,
  • restriction endonucleases. International Journal of Radiation
  • Biology, 71, 365-375.2208-2221. Goettlich, B., Reichenberger, S., Feldmann, E. and
  • Cheong, N., Okayasu, R., Shah, S., Ganguly, T., Mammen,
  • P. and Iliakis, G., 1996, In vitro rejoining of double-Pfeiffer, P., 1998, Rejoining of DNA double-strand breaks in vitro by single-strand annealing. European Journal
  • strand breaks in cellular DNA by factors present in
  • extracts of HeLa cells. International Journal of Radiationof Biochemistry, 258, 387-395. Golub, E. I., Gupta, R. C., Haaf, T., Wold, M. S. and
  • Biology, 69, 665-677.
  • Cheong, N., Perrault, A. R., Wang, H., Wachsberger, P.,Radding, C. M., 1998, Interaction of human Rad51 recombination protein with single-stranded DNA bindingMammen, P., Jackson, I. and Iliakis, G., 1999, DNA-
  • PK-independent rejoining of DNA double-strand breaksprotein, RPA. Nucleic Acids Research, 26, 5388- 5393. Haber, J. E., 1992, Exploring the pathways of homologous
  • in human cell extracts in vitro. International Journal of
  • Radiation Biology, 75, 67-81.recombination. Current Opinions in Cell Biology, 4, 401-412. Haber, J. E., 1999, DNA recombination: the replication connec-Cheong, N., Perrault, R. and Iliakis, G., 1998, In vitro
  • rejoining of DNA double strand breaks: a comparisontion. Trends in Biochemical Sciences, 24, 271-275. Hays, S. L., Firmenich, A. A., Massey, P., Banerjee, R. and
  • ofgenomic-DNAwithplasmid-DNA-basedassays.
  • International Journal of Radiation Biology, 73, 481-493.Berg, P., 1998, Studies of the interaction between Rad52 protein and the yeast single-stranded DNA binding pro-
  • Daza, P., Reichenberger, S., Goettlich, B., Hagmann, M.,
  • Feldmann, E. and Pfeiffer, P., 1996, Mechanisms oftein RPA. Molecular and Cellular Biology, 18, 4400-4406. Henner, W. D., Grunberg, S. M. and Haseltine, W. A., 1982,
  • nonhomologous DNA end-joining in frogs, mice and
  • men. Journal of Biological Chemistry, 377, 775-786.Sites and structure of g radiation-induced DNA strand breaks. Journal of Biological Chemistry, 257, 11 750-11 754.
  • DeMott, M. S., Zigman, S. and Bambara, R. A., 1998,
  • Replication Protein A Stimulates Long Patch DNA BaseHenner, W. D., Rodriguez, L. O., Hecht, S. M. and Haseltine, W. A., 1983, g ray induced deoxyribonucleic
  • Excision Repair. Journal of Biological Chemistry, 273,
  • 27492-27498.acid strand breaks. Journal of Biological Chemistry, 258, 711-713.Denko, N., Giaccia, A., Peters, B. and Stamato, T. D., 1989,
  • An asymmetric ? eld inversion gel electrophoresis methodHenricksen, L. A., Umbricht, C. B. and Wold, M. S., 1994, Recombinant replication protein A: expression, complex
  • for the separation of large DNA molecules. Analytical
  • Biochemistry, 178, 172-176.formation, and functional characterization. Journal of Biological Chemistry, 269, 11 121-11 132.Derbyshire, M. K., Epstein, L. H., Young, C. S. H., Munz,
  • P. L. and Fishel, R., 1994, Nonhomologous recombina-Iftode, C., Daniely, Y. and Borowiec, J. A., 1999, Replication protein A (RPA): the eukaryotic SSB. Critical Reviews in
  • tion in human cells. Molecular and Cellular Biology, 14,
  • 156-169.Biochemistry and Molecular Biology, 34, 141-180. Iliakis, G., 1991, The role of DNA double strand breaks in
  • DiBiase, S. J., Zeng, Z.-C., Chen, R., Hyslop, T., Curran,
  • W. J., Jr and Iliakis, G., 2000, DNA-dependent proteinionizing radiation induced killing of eukaryotic cells. BioEssays, 13, 641-648.
  • kinase stimulates an independently active, nonhomo
  • logous,end-joiningapparatus.CancerResearch,60,Iliakis, G., Mehta, R. and Jackson, M., 1992, Level of DNA double-strand break rejoining in Chinese hamster xrs-5
  • 1245-1253.
  • Dikomey, E. and Franzke, J., 1986, Three classes of DNAcells is dose-dependent: implications for the mechanism of radiosensitivity. International Journal of Radiation Biology,strand breaks induced by x-irradiation and internal b-rays.
  • International Journal of Radiation Biology, 50, 893-908.61, 315-321. Iliakis, G., Metzger, L., Denko, N. and Stamato, T. D.,
  • Fairman, M. P., Johnson, A. P. and Thacker, J., 1992, Multiple
  • components are involved in the eÝcient joining of double1991a, Detection of DNA double strand breaks in synchronous cultures of CHO cells by means of asymmetric
  • stranded DNA breaks in human cell extracts. Nucleic Acids
  • Research, 20, 4145-4152.? eld inversion gel electrophoresis. International Journal of Radiation Biology, 59, 321-341.Feldmann, E., Schmiemann, V., Goedecke, W., Reichen-
  • berger, S. and Pfeiffer, P., 2000, DNA double-strandIliakis, G. E., Cicilioni, O. and Metzger, L., 1991b, Measurement of DNA double strand breaks in CHO cells
  • break repair in cell-free extracts from Ku80-de? cient
  • cells: implications for Ku serving as an alignment factorat various stages of the cell cycle using pulsed ? eld gel electrophoresis: calibrations by means of125I decay.
  • in non-homologous DNAend-joining.NucleicAcids
  • Research, 28, 2585-2596.International Journal of Radiation Biology, 59, 343-357. Jackson, S. P. and Jeggo, P. A., 1995, DNA double-strand
  • Firmenich, A. A., Elias-Arnanz, M. and Berg, P., 1995, A
  • novel allele of Saccharomyces cerevisiae RFA1 that is de? cientbreak repair and V(D)J recombination: involvement of DNA-PK. Trends in Biochemical Sciences, 20, 412-415.
  • in recombination and repair and suppressible by RAD52.
  • Molecular and Cellular Biology, 15, 1620- 1631.Jeggo, P. A., 1990, Studies on mammalian mutants defective in rejoining double-strand breaks. Mutation Research, 239,Frankenberg-Schwager, M., 1990, Induction, repair and
  • biological relevance of radiation-induced DNA lesions1-16. Jeggo, P. A., 1998, DNA breakage and repair. Advances in
  • in eukaryotic cells. Radiation and Environmental Biophysics,
  • 29, 273-292.Genetics, 38, 186-218. Johnson, A. P. and Fairman, M. P., 1996, The identi? cationGanguly, T. and Iliakis, G., 1995, A cell-free assay using
  • cytoplasmic cell extracts to study rejoining of radiation-and characterization of mammalian proteins involved in
  • the rejoining of DNA double-strand breaks in vitro.but not for T cell antigen receptor gene recombination in vivo. Journal of Experimental Medicine, 186, 921-929.
  • Mutation Research, 364, 103-116. Painter, R. B., 1980, The role of DNA damage and repair in
  • Kanaar, R., Hoeijmakers, J. H. J. and van Gent, D. C., 1998, cell killing induced by ionizing radiation. In R. E. Meyn
  • Molecular mechanisms of DNA double-strand break and H. R. Withers (eds), Radiation Biology in Cancer Research
  • repair. Cell Biology, 8, 483-489. (New York: Raven), pp. 59-68.Kemp, L. M., Sedgwick, S. G. and Jeggo, P. A., 1984, X-ray Park, M. S., Ludwig, D. L., Stigger, E. and Lee, S.-H., 1996,
  • sensitive mutants of Chinese hamster ovary cells defective Physical interaction between human RAD52 and RPA is
  • in double-strand break rejoining. Mutation Research, 132, required for homologous recombination in mammalian
  • 189-196. cells. Journal of Biological Chemistry, 271, 18 996-19 000.
  • Kogoma, T., 1996, Recombination by replication. Cell, 85, Petes, T. D., Malone, R. E. and Symington, L. S., 1991,625-627. Recombination in yeast. In J. R. Broach J. R. Pringle
  • Labhart, P., 1999a, Ku-dependent nonhomologous DNA end-and E. W. Jones (eds), The Molecular and Cellular Biology ofjoining in Xenopus egg extracts. Molecular and Cellular the Yeast Saccharomyces (New York: Cold Spring HarborBiology, 19, 2585-2593. Laboratory Press), pp. 407-521.
  • Labhart, P., 1999b, Nonhomologous DNA end-joining in cell-Petrini, J. H. J., Bressan, D. A. and Yao, M. S., 1997, The
  • free systems. European Journal of Biochemistry, 265, 849-861. RAD52 epistasis group in mammalian double strand break
  • Lieber, M. R., Grawunder, U., Wu, X. and Yaneva, M., repair. Seminars in Immunology, 9, 181-188.
  • 1997, Tying loose ends: roles of Ku and DNA-dependent Pfeiffer, P. and Vielmetter, W., 1988, Joining of nonhomolog-
  • protein kinase in the repair of double-strand breaks. ous DNA double strand breaks in vitro. Nucleic Acids
  • Current Opinions in Genetics and Development, 7, 99-104. Research, 16, 907-924.
  • Loebrich, M., Rydberg, B. and Cooper, P. K., 1995, Repair Plug, A. W., Peters, A. H., Keegan, K. S., Hoekstra, M. F.,
  • of x-ray-induced DNA double-strand breaks in speci? c de Boer, P. and Ashley, T., 1998, Changes in protein
  • Not I restriction fragments in human ? broblasts: joining composition of meiotic nodules during mammalian mei-
  • of correct and incorrect ends. Proceedings of the National osis. Journal of Cell Science, 111, 411-423.
  • Academy of Sciences, USA, 92, 12 050-12 054. Plug, A. W., Peters, A. H., Xu, Y., Keegan, K. S., Hoekstra,
  • Mason, R. M., Thacker, J. and Fairman, M. P., 1996, The M. F., Baltimore, P., de Boer, P. and Ashley, T.,
  • joining of non-complementary DNA double-strand breaks 1997, ATM and RPA in meiotic chromosome synapsis
  • by mammalian extracts. NucleicAcidsResearch,24, and recombination. Nature Genetics, 17, 457-461.
  • 4946-4953. Resnick, M. A., 1976, The repair of double-strand breaks in
  • Metzger, L. and Iliakis, G., 1991, Kinetics of DNA double DNA: a model involving recombination. Journal of
  • strand breaks throughout the cell cycle as assayed by Theoretical Biology, 59, 97-106.
  • pulsed ? eld gel electrophoresis in CHO cells. International Rothstein, R., Michel, B. and Gangloff, S., 2000, Replication
  • Journal of Radiation Biology, 59, 1325- 1339. fork pausing and recombination or ’gimme a break‚. Genes
  • Nagelhus, T. A., Haug, T., Singh, K. K., Keshav, K. F., and Development, 14, 1-10.
  • Skorpen, F., Otterlei, M., Bharati, S., Lindmo, T., Shinohara, A. and Ogawa, T., 1995, Homologous recombina-
  • Benichou, S., Benarous, R. and Krokan, H. E., 1997, tion and the roles of double-strand breaks. Trends in
  • A Sequence in the N-terminal region of human uracil-Biological Science, 20, 387-391.
  • DNA glycosylase with homology to XPA interacts with Shinohara, A. and Ogawa, T., 1998, Stimulation by Rad52 of
  • the C-terminal part of the 34-kDa subunit of replication
  • yeastRad51-mediatedrecombination.Nature,391,
  • protein A. Journal of Biological Chemistry, 272, 6561-6566. 404-407.
  • Nevaldine, B., Longo, J. A. and Hahn, P. J., 1997, The scid Stillman, B., 1989, Initiation of eukaryotic DNA replication in
  • defect results in much slower repair of DNA double-vitro. Annual Review of Cell Biology, 5, 197-245.
  • strand breaks but not high levels of residual breaks. Sugiyama, T., Zaitseva, E. M. and Kowalczykowski, S. C.,
  • Radiation Research, 147, 535-540. 1997, A single-stranded DNA-binding protein is needed
  • New, J. H., Sugiyama, T., Zaitseva, E. and Kowalczykowski, for eÝcient presynaptic complex formation by the
  • S. C., 1998, Rad52 protein stimulates DNA strand Saccharomyces cerevisiae Rad51 protein. Journal of Biological
  • exchange by Rad51 and replication protein A. Nature, Chemistry, 272, 7940-7945.
  • 391, 407-410. Sung, P., 1997, Function of yeast Rad52 protein as a mediator
  • Nicolas, A. L. and Young, C. S. H., 1994, Characterization ofbetween replication protein A and the Rad51 recombi-
  • DNA end-joining in a mammalian cell nuclear extract: nase. Journal of Biological Chemistry, 272, 28 194-28 197.
  • junction formation is accompanied by nucleotide loss, Szostak, J. W., Orr-Weaver, T. L., Rothstein, R. J. and
  • which is limited and uniform but not site speci? c. Molecular Stahl, F. W., 1983, The double-strand-break repair
  • and Cellular Biology, 14, 170-180. model for recombination. Cell, 33, 25-35.
  • North, P., Ganesh, A. and Thacker, J., 1990, The rejoiningTeng, Y., Longhese, M. P., McDonough, G. and Waters, R.,
  • of double-strand breaks in DNA by human cell extracts.1998, Mutants with changes in diÚerent domains of yeast
  • Nucleic Acids Research, 18, 6205-6210.replication protein A exhibit diÚerences in repairing the
  • Okayasu, R. and Iliakis, G., 1993, Evidence that the productcontrol region, the transcribed strand and the non-
  • of the xrs gene is predominantly involved in the repair oftranscribed strand of the Saccharomyces cerevisiae MFA2
  • a subset of radiation-induced interphase chromosomegene. Journal of Molecular Biology, 280, 355-363.
  • breaks rejoining with fast kinetics. Radiation Research,Thacker, J., 1999, A surfeit of RAD51-like genes? Trends in
  • 138, 34-43.Genetics, 15, 166-168.
  • Ouyang, B. H., Nussenzweig, A., Kurimasa, A., da CostaThompson, L. H. and Schild, D., 1999, The contribution of
  • Soares, V., Li, X., Cordon-Cardo, C., Li, W.-h.,homologous recombination in preserving genome integ-
  • Cheong, N., Nussenzweig, M., Iliakis, G., Chen, D. J.rity in mammalian cells. Biochimie, 81, 87-105. Umezu, K., Sugawara, N., Chen, C., Haber, J. E. and
  • and Li, G. C., 1997, Ku70 is required for DNA repair
  • Kolodner, R. D., 1998, Genetic analysis of yeast RPA1tebrate cells de? cient in genes of the RAD52 epistasis group. Oncogene (in press).
  • reveals its multiple functions in DNA metabolism. Genetics,
  • 148, 989-1005.Ward, J. F., 1990, The yield of DNA double-strand breaks produced intracellularly by ionizing radiation: a review.Wachsberger, P. R., Li, W.-H., Guo, M., Chen, D., Cheong,
  • N., Ling, C. C. and Iliakis, G., 1999, Rejoining of DNAInternational Journal of Radiation Biology, 57, 1141- 1150. Weaver, D. T., 1996, Regulation and repair of double-strand
  • double strand breaks in Ku80-de? cient mouse ? broblasts.
  • Radiation Research, 151, 398-407.DNA breaks. Critical Reviews in Eukaryotic Gene Expression, 6, 345-375.
  • Wang, H., Zeng, Z.-C., Bui, T.-A., DiBiase, S. J., Qin, W.,
  • Xia, F., Powell, S. N. and Iliakis, G., 2001, Non-Weibezahn, K. F. and Coquerelle, T., 1981, Radiation induced DNA double strand breaks are rejoined by
  • homologous end-joining of ionizing radiation-induced
  • DNA double stranded breaks in human tumor cellsligationandrecombinationprocesses.NucleicAcids Research, 3139-3150.
  • de? cient in BRCA1 or BRCA2. Cancer Research, 61,
  • 270-277.Wold, M. S., 1997, Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukary-Wang, H., Zeng, Z.-C., Bui, T.-A., Sonoda, E., Takata, M.,
  • Takeda, S. and Iliakis, G., 2000, EÝcient rejoining ofotic DNA metabolism. Annual Reviews in Biochemistry, 66, 61-92.
  • Takeda, S. and Iliakis, G., 2000, EÝcient rejoining ofotic DNA metabolism. Annual Reviews in Biochemistry, 66, 61-92.

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