The structural basis for discrimination of DNA substrates by Cre recombinase will be investigated. Cre recombinase promotes a specific crossing-over reaction between two 34 basepair loxP DNA sequences in vitro and in vivo. The Cre-lox system has shown great utility for generating specific chromosomal rearrangements in living cells and organisms. Its greatest limitation is the requirement for introducing lox sequences into the target genomes by relatively inefficient methods. Although lox-related sequences (LRSs) occur in mammalian genomes, they do not function efficiently enough for most applications. If the structural mechanisms for discriminating IoxP sequences from other DNA can be identified, they could be redirected by protein engineering to allow Cre to recognize existing genomic LRSs and non-lox sequences. The large size of the lox sequence, the multimeric active complex, and multi-step reaction allow for several levels of substrate discrimination. To define the sequence requirements for efficient recombination of the loxP site at the nucleotide level, the recombination activity of a systematic set of singly and doubly substituted loxP sequences will be quantitatively assessed. For low activity substrates, the reaction step(s) that is(are) involved in discrimination will be determined by analysis of DNA binding, active complex assembly and catalysis. To understand the structural basis for inactivity of substrates which are bound but do not undergo recombination, X-ray crystal structures of the impaired Cre-variant lox complexes wilt be analyzed for structural differences from the cognate loxP complexes. The role of an eight nucleotide region of LoxP that separates the two Cre binding sites in promoting efficient recombination will be ascertained from reaction intermediate structures incorporating this region. These structures wilt also act as references for comparisons with the mutant complexes. Since the Cre-lox reaction is paradigmatic for site-specific recombination, these results of this work should be applicable to understanding control of specificity by other tyrosine recombinases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Biochemistry Study Section (BIO)
Program Officer
Portnoy, Matthew
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of California Davis
Anatomy/Cell Biology
Schools of Arts and Sciences
United States
Zip Code
Habrian, Chris; Chandrasekhara, Adithi; Shahrvini, Bita et al. (2016) Inhibition of Escherichia coli CTP Synthetase by NADH and Other Nicotinamides and Their Mutual Interactions with CTP and GTP. Biochemistry 55:5554-5565
Gelato, Kathy A; Martin, Shelley S; Liu, Patty H et al. (2008) Spatially directed assembly of a heterotetrameric Cre-Lox synapse restricts recombination specificity. J Mol Biol 378:653-65
Gelato, Kathy A; Martin, Shelley S; Wong, Scott et al. (2006) Multiple levels of affinity-dependent DNA discrimination in Cre-LoxP recombination. Biochemistry 45:12216-26
Endrizzi, James A; Kim, Hanseong; Anderson, Paul M et al. (2005) Mechanisms of product feedback regulation and drug resistance in cytidine triphosphate synthetases from the structure of a CTP-inhibited complex. Biochemistry 44:13491-9
Gelato, Kathy A; Martin, Shelley S; Baldwin, Enoch P (2005) Reversed DNA strand cleavage specificity in initiation of Cre-LoxP recombination induced by the His289Ala active-site substitution. J Mol Biol 354:233-45
Endrizzi, James A; Kim, Hanseong; Anderson, Paul M et al. (2004) Crystal structure of Escherichia coli cytidine triphosphate synthetase, a nucleotide-regulated glutamine amidotransferase/ATP-dependent amidoligase fusion protein and homologue of anticancer and antiparasitic drug targets. Biochemistry 43:6447-63
Martin, Shelley S; Chu, Victor C; Baldwin, Enoch (2003) Modulation of the active complex assembly and turnover rate by protein-DNA interactions in Cre-LoxP recombination. Biochemistry 42:6814-26