Abstract
With improvements in biophysical approaches, there is growing interest in characterizing large, flexible multi-protein complexes. The use of recombinant baculoviruses to express heterologous genes in cultured insect cells has advantages for the expression of human protein complexes because of the ease of co-expressing multiple proteins in insect cells and the presence of a conserved post-translational machinery that introduces many of the same modifications found in human cells. Here we describe the preparation of recombinant baculoviruses expressing DNA ligase IIIα, XRCC1, and TDP1, their subsequent co-expression in cultured insect cells, the purification of complexes containing DNA ligase IIIα from insect cell lysates, and their characterization by multi-angle light scattering linked to size exclusion chromatography and negative stain electron microscopy.
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References
Abdella R, Talyzina A, Chen S, Inouye CJ, Tjian R, He Y (2021) Structure of the human mediator-bound transcription preinitiation complex. Science 372(6537):52–56
Chen S, Lee L, Naila T, Fishbain S, Wang A, Tomkinson AE, Lees-Miller SP, He Y (2021) Structural basis of long-range to short-range synapsis in NHEJ. Nature 593(7858):294–298
Hammel M, Yu Y, Mahaney BL, Cai B, Ye R, Phipps BM, Rambo RP, Hura GL, Pelikan M, So S, Abolfath RM, Chen DJ, Lees-Miller SP, Tainer JA (2010) Ku and DNA-dependent protein kinase dynamic conformations and assembly regulate DNA binding and the initial non-homologous end joining complex. J Biol Chem 285:1414–1423
Hammel M, Yu Y, Radhakrishnan SK, Chokshi C, Tsai MS, Matsumoto Y, Kuzdovich M, Remesh SG, Fang S, Tomkinson AE, Lees-Miller SP, Tainer JA (2016) An intrinsically disordered APLF links Ku, DNA-PKcs, and XRCC4-DNA ligase IV in an extended flexible non-homologous end joining complex. J Biol Chem 291:26987–27006
Hammel M, Rashid I, Sverzhinsky A, Pourfarjam Y, Tsai MS, Ellenberger T, Pascal JM, Kim IK, Tainer JA, Tomkinson AE (2021) An atypical BRCT-BRCT interaction with the XRCC1 scaffold protein compacts human DNA ligase IIIalpha within a flexible DNA repair complex. Nucleic Acids Res 49:306–321
Schneidman-Duhovny D, Hammel M, Tainer JA, Sali A (2016) FoXS, FoXSDock and MultiFoXS: single-state and multi-state structural modeling of proteins and their complexes based on SAXS profiles. Nucleic Acids Res 44:W424–W429
Ellenberger T, Tomkinson AE (2008) Eukaryotic DNA ligases: structural and functional insights. Annu Rev Biochem 77:313–338
Lakshmipathy U, Campbell C (1999) The human DNA ligase III gene encodes nuclear and mitochondrial proteins. Mol Cell Biol 19:3869–3876
Caldecott KW, McKeown CK, Tucker JD, Ljungquist S, Thompson LH (1994) An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III. Mol Cell Biol 14:68–76
Caldecott KW (2019) XRCC1 protein; form and function. DNA Repair (Amst) 81:102664
Gao Y, Katyal S, Lee Y, Zhao J, Rehg JE, Russell HR, McKinnon PJ (2011) DNA ligase III is critical for mtDNA integrity but not Xrcc1-mediated nuclear DNA repair. Nature 471:240–244
Simsek D, Furda A, Gao Y, Artus J, Brunet E, Hadjantonakis AK, Van Houten B, Shuman S, McKinnon PJ, Jasin M (2011) Crucial role for DNA ligase III in mitochondria but not in Xrcc1-dependent repair. Nature 471:245–248
Le Chalony C, Hoffschir F, Gauthier LR, Gross J, Biard DS, Boussin FD, Pennaneach V (2012) Partial complementation of a DNA ligase I deficiency by DNA ligase III and its impact on cell survival and telomere stability in mammalian cells. Cell Mol Life Sci 69:2933–2949
Lakshmipathy U, Campbell C (2000) Mitochondrial DNA ligase III function is independent of Xrcc1. Nucleic Acids Res 28:3880–3886
Huang SY, Murai J, Dalla Rosa I, Dexheimer TS, Naumova A, Gmeiner WH, Pommier Y (2013) TDP1 repairs nuclear and mitochondrial DNA damage induced by chain-terminating anticancer and antiviral nucleoside analogs. Nucleic Acids Res 41:7793–7803
El-Khamisy SF, Saifi GM, Weinfeld M, Johansson F, Helleday T, Lupski JR, Caldecott KW (2005) Defective DNA single-strand break repair in spinocerebellar ataxia with axonal neuropathy-1. Nature 434:108–113
Della-Maria J, Zhou Y, Tsai MS, Kuhnlein J, Carney JP, Paull TT, Tomkinson AE (2011) Human Mre11/human Rad50/Nbs1 and DNA ligase IIIalpha/XRCC1 protein complexes act together in an alternative nonhomologous end joining pathway. J Biol Chem 286:33845–33853
Cannan WJ, Rashid I, Tomkinson AE, Wallace SS, Pederson DS (2017) The human ligase IIIalpha-XRCC1 protein complex performs DNA Nick repair after transient unwrapping of nucleosomal DNA. J Biol Chem 292:5227–5238
Slotboom DJ, Duurkens RH, Olieman K, Erkens GB (2008) Static light scattering to characterize membrane proteins in detergent solution. Methods 46:73–82
Wen J, Arakawa T, Philo JS (1996) Size-exclusion chromatography with on-line light-scattering, absorbance, and refractive index detectors for studying proteins and their interactions. Anal Biochem 240:155–166
Sverzhinsky A, Fabre L, Cottreau AL, Biot-Pelletier DM, Khalil S, Bostina M, Rouiller I, Coulton JW (2014) Coordinated rearrangements between cytoplasmic and periplasmic domains of the membrane protein complex ExbB-ExbD of Escherichia coli. Structure 22:791–797
Cheng Y, Grigorieff N, Penczek PA, Walz T (2015) A primer to single-particle cryo-electron microscopy. Cell 161:438–449
Ohi M, Li Y, Cheng Y, Walz T (2004) Negative staining and image classification - powerful tools in modern electron microscopy. Biol Proced Online 6:23–34
Scarff CA, Fuller MJG, Thompson RF, Iadanza, MG (2018) Variations on negative stain electron microscopy methods: tools for tackling challenging systems. J Vis Exp (132):57199
Gallagher JR, Kim AJ, Gulati NM, Harris AK (2019) Negative-stain transmission electron microscopy of molecular complexes for image analysis by 2D class averaging. Curr Protoc Microbiol 54:e90
Vijayachandran LS, Viola C, Garzoni F, Trowitzsch S, Bieniossek C, Chaillet M, Schaffitzel C, Busso D, Romier C, Poterszman A, Richmond TJ, Berger I (2011) Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells. J Struct Biol 175:198–208
Gradia SD, Ishida JP, Tsai MS, Jeans C, Tainer JA, Fuss JO (2017) MacroBac: new technologies for robust and efficient large-scale production of recombinant multiprotein complexes. Methods Enzymol 592:1–26
Acknowledgments
This work was supported in part by National Institutes of Health (NIH) grants R01 ES012512 (A.E.T.) and a Discovery grant RGPIN-2015-05776 from the Natural Science and Engineering Research Council of Canada (J.M.P.). The collaboration between the Tomkinson and Pascal laboratories and the Expression and Molecular Biology Core led by Tsai was supported by the Structural Cell Biology of DNA Repair Program (P01 CA92584). AET acknowledges support from the University of New Mexico Comprehensive Cancer Center.
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Rashid, I. et al. (2022). Purification and Characterization of Human DNA Ligase IIIα Complexes After Expression in Insect Cells. In: Mosammaparast, N. (eds) DNA Damage Responses. Methods in Molecular Biology, vol 2444. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2063-2_15
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DOI: https://doi.org/10.1007/978-1-0716-2063-2_15
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