Abstract
Affinity pulldown is a powerful technique to discover novel interaction partners and verify a predicted physical association between two or more proteins. Pulldown assays capture a target protein fused with an affinity tag and analyze the complexed proteins. Here, we detail methods of pulldown assays for two high-affinity peptide fusion tags, Flag tag (DYKDDDDK) and hexahistidine tag (6xHis), to study protein–protein interactions of human NEIL1 glycosylase and the checkpoint protein complex RAD9–RAD1–HUS1 (9-1-1). We uncover unique interactions between 9-1-1 and NEIL1, which suggest a possible inhibitory role of the disordered, phosphorylated C-terminal region of RAD9 in regulating NEIL1 activity in base excision repair through lack of physical association of 9-1-1 and NEIL1.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Dutta A, Yang C, Sengupta S, Mitra S, Hegde ML (2015) New paradigms in the repair of oxidative damage in human genome: mechanisms ensuring repair of mutagenic base lesions during replication and involvement of accessory proteins. Cell Mol Life Sci 72:1679–1698
Meyerkord CL, Fu H (eds) (2015) Protein-proteininteractions. In Methods and applications. Methods in molecular biology
Guan X, Bai H, Shi G, Theriot CA, Hazra TK, Mitra S et al (2007) The human checkpoint sensor Rad9-Rad1-Hus1 interacts with and stimulates NEIL1 glycosylase. Nucleic Acids Res 35:2463–2472
Balakrishnan L, Brandt PD, Lindsey-Boltz LA, Sancar A, Bambara RA (2009) Long patch base excision repair proceeds via coordinated stimulation of the multienzyme DNA repair complex. J Biol Chem 284:15158–15172
Panigrahi SK, Hopkins KM, Lieberman HB (2015) Regulation of NEIL1 protein abundance by RAD9 is important for efficient base excision repair. Nucleic Acids Res 43:4531–4546
Hazra TK, Mitra S (2006) Purification and characterization of NEIL1 and NEIL2, members of a distinct family of mammalian DNA glycosylases for repair of oxidized bases. Methods Enzymol 408:33–48
Bermudez VP, Lindsey-Boltz LA, Cesare AJ, Maniwa Y, Griffith JD, Hurwitz J et al (2003) Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro. Proc Natl Acad Sci U S A 100:1633–1638
Querol-Audi J, Yan C, Xu X, Tsutakawa SE, Tsai MS, Tainer JA et al (2012) Repair complexes of FEN1 endonuclease, DNA, and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability. Proc Natl Acad Sci U S A 109:8528–8533
Singh VK, Nurmohamed S, Davey SK, Jia Z (2007) Tri-cistronic cloning, overexpression and purification of human Rad9, Rad1, Hus1 protein complex. Protein Expr Purif 54:204–211
Acknowledgements
We thank Dr. Priscilla K. Cooper (Lawrence Berkeley National Laboratory, Berkeley, California) for her scientific guidance and the Expression and Molecular Biology Core staff Khanh K. Nguyen, Aye Su Hlaing, Phyo S. Aung, and Ahmed M. Akbar for their technical assistance in preparing both full-length and C-terminally truncated human 9-1-1 trimeric proteins from bacteria and insect cells. We thank Dr. Vinay Kumar Singh from the Zongchao Jia’s lab (Queen’s University, Kinston, Ontario, Canada) for providing the E. coli tricistronic expression vector for full-length human 9-1-1 and Dr. Vladimir P. Bermudez from the Hurwitz lab (Memorial Sloan-Kettering Cancer Institute, New York, New York) for their generous gifts of the baculovirus transfer vectors, baculovirus stocks, and in-house rabbit polyclonal antibodies for RAD9, RAD1, and HUS1 and Drs. Muralidhar L. Hegde and Sankar Mitra (Houston Methodist Cancer Center, Houston Methodist Research Institute, Huston, Texas) for providing purified NEIL1 protein. This work was supported by the National Cancer Institute/National Institutes of Health (NCI/NIH) program project grant P01 CA092584 for the Structural Cell Biology of DNA Repair Machines (SBDR) to all authors DTM, PSW, JMJ, and MST.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
McDonald, D.T., Wang, P.S., Moitoza Johnson, J., Tsai, MS. (2023). Using Affinity Pulldown Assays to Study Protein–Protein Interactions of Human NEIL1 Glycosylase and the Checkpoint Protein RAD9–RAD1–HUS1 (9-1-1) Complex. In: Bhakat, K.K., Hazra, T.K. (eds) Base Excision Repair Pathway. Methods in Molecular Biology, vol 2701. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3373-1_13
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3373-1_13
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3372-4
Online ISBN: 978-1-0716-3373-1
eBook Packages: Springer Protocols