Poster abstracts

Poster number 37 submitted by Jonathan Montgomery

Dynamic Conformational Changes Modulate Interactions between Cre Recombinase and DNA Substrates

Jonathan S. Montgomery (The Ohio State University), Mark P. Foster (The Ohio State University)

Abstract:
Cre (Causes Recombination) is a site-specific DNA recombinase and promising gene editing tool, that manipulates genetic material without inducing cytotoxic double-strand DNA breaks1. This is a central advantage over other DNA editing enzymes, such as Cas9 and ZFN’s, 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.

Nuclear Magnetic Resonance structural probes and dynamics have identified that Cre adopts an autoinhibited conformation in the absence of DNA, 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 autoinhibition functions to modulate DNA binding by exchanging between binding competent and incompetent conformations. Nuclear Magnetic Resonance (NMR) dynamics experiments, particularly chemical exchange saturation transfer (CEST), were able to quantify this dynamic conformational selection. Together with additional biophysical measurements, we can determine the biological role of this autoinhibitory conformation.

References:
1. Ghosh et al. Cre–loxP biochemistry. Methods 28, 374–383 (2002).
2. He et al. Tissue-Specific Targeting of the Pthrp Gene: The Generation of Mice with Floxed Alleles*. Endocrinology 142, 2070–2077 (2001).
3. Turan et al. Recombinase-Mediated Cassette Exchange (RMCE): Traditional Concepts and Current Challenges. JMB 407, 193-221 (2011).
4. Xie et al. Off-Target Deletion of Conditional Dbc1 Allele in the Foxp3YFP-Cre Mouse Line under Specific Setting. Cells 8, 1309 (2019).
5. Unnikrishnan et al. DNA binding induces a cis -to- trans switch in Cre recombinase to enable intasome assembly. PNAS 117, 24849–24858 (2020).
6. Stachowski et al. Mechanisms of Cre recombinase synaptic complex assembly and activation illuminated by Cryo-EM. NAR 50, 1753–1769 (2022).

Keywords: Autoinhibition, NMR Dynamics, Cre Recombinase