Functional mapping of Cre recombinase by pentapeptide insertional mutagenesis.

The Journal of Biological Chemistry
Vladislav PetyukBrian Sauer

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 r...Continue Reading

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Feb 10, 2009·ACS Chemical Biology·Kelly A DaggettT Ashton Cropp
Jun 21, 2011·Plant Science : an International Journal of Experimental Plant Biology·Hong MaYuanlei Hu
Mar 16, 2016·Proceedings of the National Academy of Sciences of the United States of America·Fantao MengQi Wu
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Dec 25, 2021·Nucleic Acids Research·Jenna HoerstenFrank Buchholz

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