Functional mapping of Cre recombinase by pentapeptide insertional mutagenesis - PubMed (original) (raw)
. 2004 Aug 27;279(35):37040-8.
doi: 10.1074/jbc.M406042200. Epub 2004 Jun 24.
Affiliations
- PMID: 15218019
- DOI: 10.1074/jbc.M406042200
Free article
Functional mapping of Cre recombinase by pentapeptide insertional mutagenesis
Vladislav Petyuk et al. J Biol Chem. 2004.
Free article
Abstract
Cre is a site-specific recombinase from bacteriophage P1. It is a member of the tyrosine integrase family and catalyzes reciprocal recombination between specific 34-bp sites called loxP. To analyze the structure-function relationships of this enzyme, we performed large scale pentapeptide insertional mutagenesis to generate insertions of five amino acids at random positions in the protein. The high density of insertion mutations into Cre allowed us to identify an unexpected degree of functional tolerance to insertions into the 4-5 beta-hairpin and into the loop between helices J and K (both of which contact the DNA in the minor groove) and also into helix A. The phenotypes of the majority of inserts allowed us to confirm a variety of predictions made on the basis of sequence conservation, known three-dimensional structure, and proposed catalytic mechanism. In particular, most insertions into conserved regions or secondary structure elements inactivated Cre, and most insertions located in nonconserved, unstructured regions preserved Cre activity. Less expectedly, the non-conserved and poorly structured loops and linkers between helices A-B, E-F, and M-N did not tolerate insertions, thus identifying these as critical regions for recombinase activity. We purified and characterized in vitro several representatives of these "unexpected" Cre insertion mutants. The role of those regions in the recombination process is discussed.
Similar articles
- Structure-function correlations in the XerD site-specific recombinase revealed by pentapeptide scanning mutagenesis.
Cao Y, Hallet B, Sherratt DJ, Hayes F. Cao Y, et al. J Mol Biol. 1997 Nov 21;274(1):39-53. doi: 10.1006/jmbi.1997.1380. J Mol Biol. 1997. PMID: 9398514 - [Progress in the study of the structure and function of Cre recombinase].
Wang LX, Wang Y, Hu YL, Gao Y, Lin ZP. Wang LX, et al. Sheng Wu Gong Cheng Xue Bao. 2002 Sep;18(5):531-5. Sheng Wu Gong Cheng Xue Bao. 2002. PMID: 12561193 Review. Chinese. - Identification of Cre residues involved in synapsis, isomerization, and catalysis.
Lee L, Sadowski PD. Lee L, et al. J Biol Chem. 2003 Sep 19;278(38):36905-15. doi: 10.1074/jbc.M305464200. Epub 2003 Jul 8. J Biol Chem. 2003. PMID: 12851389 - Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse.
Guo F, Gopaul DN, van Duyne GD. Guo F, et al. Nature. 1997 Sep 4;389(6646):40-6. doi: 10.1038/37925. Nature. 1997. PMID: 9288963 - A structural view of cre-loxp site-specific recombination.
Van Duyne GD. Van Duyne GD. Annu Rev Biophys Biomol Struct. 2001;30:87-104. doi: 10.1146/annurev.biophys.30.1.87. Annu Rev Biophys Biomol Struct. 2001. PMID: 11340053 Review.
Cited by
- Activation of recombinases at specific DNA loci by zinc-finger domain insertions.
Mukhametzyanova L, Schmitt LT, Torres-Rivera J, Rojo-Romanos T, Lansing F, Paszkowski-Rogacz M, Hollak H, Brux M, Augsburg M, Schneider PM, Buchholz F. Mukhametzyanova L, et al. Nat Biotechnol. 2024 Jan 31. doi: 10.1038/s41587-023-02121-y. Online ahead of print. Nat Biotechnol. 2024. PMID: 38297187 - Systematic dissection of key factors governing recombination outcomes by GCE-SCRaMbLE.
Zhang H, Fu X, Gong X, Wang Y, Zhang H, Zhao Y, Shen Y. Zhang H, et al. Nat Commun. 2022 Oct 3;13(1):5836. doi: 10.1038/s41467-022-33606-0. Nat Commun. 2022. PMID: 36192484 Free PMC article. - Pairing of single mutations yields obligate Cre-type site-specific recombinases.
Hoersten J, Ruiz-Gómez G, Lansing F, Rojo-Romanos T, Schmitt LT, Sonntag J, Pisabarro MT, Buchholz F. Hoersten J, et al. Nucleic Acids Res. 2022 Jan 25;50(2):1174-1186. doi: 10.1093/nar/gkab1240. Nucleic Acids Res. 2022. PMID: 34951450 Free PMC article. - Intein-mediated Cre protein assembly for transgene excision in hybrid progeny of transgenic Arabidopsis.
Ge J, Wang L, Yang C, Ran L, Wen M, Fu X, Fan D, Luo K. Ge J, et al. Plant Cell Rep. 2016 Oct;35(10):2045-53. doi: 10.1007/s00299-016-2015-x. Epub 2016 Jun 20. Plant Cell Rep. 2016. PMID: 27324752 - New inducible genetic method reveals critical roles of GABA in the control of feeding and metabolism.
Meng F, Han Y, Srisai D, Belakhov V, Farias M, Xu Y, Palmiter RD, Baasov T, Wu Q. Meng F, et al. Proc Natl Acad Sci U S A. 2016 Mar 29;113(13):3645-50. doi: 10.1073/pnas.1602049113. Epub 2016 Mar 14. Proc Natl Acad Sci U S A. 2016. PMID: 26976589 Free PMC article.
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources