dc.contributor.author | Lindenburg, LH | |
dc.contributor.author | Pantelejevs, T | |
dc.contributor.author | Gielen, F | |
dc.contributor.author | Zuazua-Villar, P | |
dc.contributor.author | Butz, M | |
dc.contributor.author | Rees, E | |
dc.contributor.author | Kaminski, CF | |
dc.contributor.author | Downs, JA | |
dc.contributor.author | Hyvönen, M | |
dc.contributor.author | Hollfelder, F | |
dc.date.accessioned | 2022-10-28T15:30:57Z | |
dc.date.issued | 2021-11-11 | |
dc.date.updated | 2022-10-28T14:47:55Z | |
dc.description.abstract | Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled module combinations were stronger binders than any of the module combinations in the natural repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with a weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by -2.4 kCal/mol compared to the strongest natural repeat, BRC4. A crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended β-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear RAD51 foci after ionizing radiation. | en_GB |
dc.description.sponsorship | Biotechnology and Biological Sciences Research Council | en_GB |
dc.description.sponsorship | European Research Council | en_GB |
dc.description.sponsorship | Marie Curie Research Grant | en_GB |
dc.description.sponsorship | Cancer Research UK | en_GB |
dc.description.sponsorship | Engineering and Physical Sciences Research Council | en_GB |
dc.description.sponsorship | Engineering and Physical Sciences Research Council | en_GB |
dc.description.sponsorship | Wellcome Trust | en_GB |
dc.description.sponsorship | Wellcome Trust | en_GB |
dc.description.sponsorship | Medical Research Council | en_GB |
dc.description.sponsorship | Medical Research Council | en_GB |
dc.description.sponsorship | Schweizerischer Nationalfonds | en_GB |
dc.format.extent | e2017708118- | |
dc.format.medium | Print | |
dc.identifier.citation | Vol. 118, No. 46, article e2017708118 | en_GB |
dc.identifier.doi | https://doi.org/10.1073/pnas.2017708118 | |
dc.identifier.grantnumber | BB/K013629/1 | en_GB |
dc.identifier.grantnumber | 695669 | en_GB |
dc.identifier.grantnumber | 659029 | en_GB |
dc.identifier.grantnumber | C7905/A25715 | en_GB |
dc.identifier.grantnumber | EP/L015889/1 | en_GB |
dc.identifier.grantnumber | EP/H018301/1 | en_GB |
dc.identifier.grantnumber | 3-3249/Z/16/Z | en_GB |
dc.identifier.grantnumber | 089703/Z/09/Z | en_GB |
dc.identifier.grantnumber | MR/K015850/1 | en_GB |
dc.identifier.grantnumber | MR/K02292X/1 | en_GB |
dc.identifier.uri | http://hdl.handle.net/10871/131488 | |
dc.identifier | ORCID: 0000-0003-0604-7224 (Gielen, Fabrice) | |
dc.language.iso | en | en_GB |
dc.publisher | National Academy of Sciences | en_GB |
dc.relation.url | https://www.ncbi.nlm.nih.gov/pubmed/34772801 | en_GB |
dc.relation.url | https://github.com/quantitativeimaging/icetropy | en_GB |
dc.rights | © 2021. Published under the PNAS license. | en_GB |
dc.subject | BRC repeats | en_GB |
dc.subject | RAD51 | en_GB |
dc.subject | modular protein engineering | en_GB |
dc.subject | protein evolution | en_GB |
dc.subject | synthetic biology | en_GB |
dc.subject | Amino Acid Sequence | en_GB |
dc.subject | BRCA2 Protein | en_GB |
dc.subject | Cell Line, Tumor | en_GB |
dc.subject | Humans | en_GB |
dc.subject | Protein Binding | en_GB |
dc.subject | Rad51 Recombinase | en_GB |
dc.title | Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats. | en_GB |
dc.type | Article | en_GB |
dc.date.available | 2022-10-28T15:30:57Z | |
dc.identifier.issn | 0027-8424 | |
exeter.article-number | ARTN e2017708118 | |
exeter.place-of-publication | United States | |
dc.description | This is the final version. Available from the National Academy of Sciences via the DOI in this record. | en_GB |
dc.description | SI Appendix contains detailed descriptions of the cloning of bacterial expression constructs for the 64 shuffled BRC peptide variants, cloning of mammalian expression constructs, and notes on the soluble expression of the shuffled BRC peptide variants. Also included is a description of ITC used to cross-validate the microfluidic measurements, single concentration point measurements carried out with microfluidics, and exemplary titrations carried out by microfluidics. The fluorescence anisotropy data obtained for the 64 separate titrations as well as the Matlab script used in the analysis have been uploaded as separate files. The supplementary data also contain an analysis on the effect of shuffling of BRC peptides and in particular on the effect of the exact shuffle cutoff point placement. X-ray crystallography electron density map images, data collection, and refinement statistics are also to be found in SI Appendix. Additional cell images highlighting the pan-nuclear signal of RAD51 are also included in SI Appendix. The coordinates and corresponding structure factors for the monomeric RAD51:BRC8-2 complex have been deposited to the PDB under accession code 6HQU. As described previously (49), the transformation from intensity maps into anisotropy values from image data was carried out with a custom Matlab code available on GitHub (https://github.com/quantitativeimaging/icetropy). A custom Matlab script used to fit Kd values for the unlabeled competitive GB1-BRC peptides can be found in SI Appendix, Datasets S1–S4. All other study data are included in the article and/or supporting information. | en_GB |
dc.identifier.eissn | 1091-6490 | |
dc.identifier.journal | Proceedings of the National Academy of Sciences | en_GB |
dc.relation.ispartof | Proc Natl Acad Sci U S A, 118(46) | |
dc.rights.uri | http://www.rioxx.net/licenses/all-rights-reserved | en_GB |
dcterms.dateAccepted | 2021-08-10 | |
rioxxterms.version | AM | en_GB |
rioxxterms.licenseref.startdate | 2021-11-11 | |
rioxxterms.type | Journal Article/Review | en_GB |
refterms.dateFCD | 2022-10-28T15:24:01Z | |
refterms.versionFCD | AM | |
refterms.dateFOA | 2022-10-28T15:31:01Z | |
refterms.panel | B | en_GB |
refterms.dateFirstOnline | 2021-11-11 | |