Poster abstracts

Poster number 115 submitted by Jonathan Montgomery

Mechanism of Selective DNA Binding by Cre Recombinase

Jonathan S. Montgomery (Ohio State Biochemistry Program), Mark P. Foster (Department of Chemistry and Biochemistry)

Abstract:
Cre (Causes Recombination) is a site-specific DNA recombinase and promising gene editing tool, allowing for manipulation of genetic material without inducing cytotoxic double-strand DNA breaks1. This is a central advantage over other DNA editing enzymes as double-strand DNA breaks are targeted by inefficient and error-prone host repair pathways. Cre has seen extensive use in the study of human health and disease through the production of conditional knockout mice and insertion of target genes through recombinase mediated cassette exchange (RMCE)2,3. However, its use as a therapeutic in human health has been hindered by a lack of understanding of the site-selection process and off-target recombination has been observed in vivo4. Recent evidence suggests that in the absence of DNA Cre adopts an auto-inhibited conformation, in which the C-terminus docks over the active and DNA binding sites. Upon binding DNA, the C-terminus is extended and contributes to cooperative binding in higher order oligomers5,6. We hypothesize that auto-inhibition functions to modulate DNA binding specificity by exchanging between binding competent and incompetent conformations. We have employed an integrated biophysical approach using isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) to study the mechanism by which Cre selectively binds cognate loxP sites. We illustrate that the presence of a binding-incompetent, auto-inhibited state modulates the affinity of Cre towards loxP and non-cognate substrates, and that selective binding to loxP sites is achieved in part by conformational changes that are not present in non-cognate complexes.

References:
(1) Ghosh, K., et al. Cre–LoxP Biochemistry. Methods 2002, 28 (3), 374–383.
(2) He, B., et al. Tissue-Specific Targeting of the Pthrp Gene: The Generation of Mice with Floxed Alleles*. Endocrinology 2001, 142 (5), 2070–2077.
(3) Turan, S., et al. Recombinase-Mediated Cassette Exchange (RMCE): Traditional Concepts and Current Challenges JMB 2011, 407, 193-221.
(4) Xie, C., et al. Off-Target Deletion of Conditional Dbc1 Allele in the Foxp3YFP-Cre Mouse Line under Specific Setting. Cells 2019, 8 (11), 1309.
(5) Unnikrishnan, A., et al. DNA Binding Induces a Cis -to- Trans Switch in Cre Recombinase to Enable Intasome Assembly. Proc Natl Acad Sci USA 2020, 117 (40), 24849–24858.
(6) Stachowski, K., et al. Mechanisms of Cre Recombinase Synaptic Complex Assembly and Activation Illuminated by Cryo-EM. Nucleic Acids Research 2022, 50 (3), 1753–1769.

Keywords: DNA Binding Specificity, Auto-inhibition, Protein Dynamics