Poster abstracts
Poster number 88 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 that can manipulate genetic material at cognate loxP sequences. Recombination proceeds through a Holliday junction intermediate and generates intact recombinant products1, in contrast to other common gene editing technologies such as Cas9 and zinc finger nucleases that produce cytotoxic double-strand breaks2. This is a central advantage of the Cre system for biomedical applications as double-strand DNA breaks are targeted by inefficient and error-prone host repair pathways. Cre has been used extensively 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)3,4. However, its use as a therapeutic in human health has been hindered by a lack of understanding of the site-selection process, as off-target recombination has been observed in vivo5.
Recent nuclear magnetic resonance spectroscopy (NMR) experiments identified an unexpected autoinhibited conformation in which the C-terminus of the apo enzyme docks over the active and DNA binding sites. Upon binding DNA, the C-terminus is extended and contributes to cooperative binding and assembly of recombinant tetrameric complexes6. We hypothesize that autoinhibition modulates DNA binding affinity by exchanging between binding competent and incompetent conformations. NMR relaxation experiments, particularly chemical exchange saturation transfer (CEST), were used to quantify this dynamic conformational selection process. Together with additional biophysical measurements, these experiments advance our understanding of how Cre achieves specificity towards its cognate DNA substrates.
References:
1.Ghosh, K, et. al., Cre–loxP biochemistry. Methods 28, 374–383 (2002).
2.Jiang, F, et. al. CRISPR–Cas9 Structures and Mechanisms. Annu. Rev. Biophys. 46, 505–529 (2017).
3.He, B. et al. Tissue-Specific Targeting of the Pthrp Gene: The Generation of Mice with Floxed Alleles*. Endocrinology 142, 2070–2077 (2001).
4.Suran, T. et. al. Recombinase-Mediated Cassette Exchange (RMCE): Traditional Concepts and Current Challenges. JMB 407, 193-221 (2011).
5.Xie, C. et al. Off-Target Deletion of Conditional Dbc1 Allele in the Foxp3YFP-Cre Mouse Line under Specific Setting. Cells 8, 1309 (2019).
6.Unnikrishnan, A. et al. DNA binding induces a cis -to- trans switch in Cre recombinase to enable intasome assembly. PNAS 117, 24849–24858 (2020).
Keywords: NMR Dynamics, Protein-DNA interactions, Biophysics