Solution properties of the archaeal CRISPR DNA repeat-binding homeodomain protein Cbp2

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) form the basis of diverse adaptive immune systems directed primarily against invading genetic elements of archaea and bacteria. Cbp1 of the crenarchaeal thermoacidophilic order Sulfolobales, carrying three imperfect repeats, binds specifically to CRISPR DNA repeats and has been implicated in facilitating production of long transcripts from CRISPR loci. Here, a second related class of CRISPR DNA repeat-binding protein, denoted Cbp2, is characterized that contains two imperfect repeats and is found amongst members of the crenarchaeal thermoneutrophilic order Desulfurococcales. DNA repeat-binding properties of the Hyperthermus butylicus protein Cbp2Hb were characterized and its three-dimensional structure was determined by NMR spectroscopy. The two repeats generate helix-turn-helix structures separated by a basic linker that is implicated in facilitating high affinity DNA binding of Cbp2 by tethering the two domains. Structural studies on mutant proteins provide support for Cys7 and Cys28 enhancing high thermal stability of Cbp2Hb through disulphide bridge formation. Consistent with their proposed CRISPR transcriptional regulatory role, Cbp2Hb and, by inference, other Cbp1 and Cbp2 proteins are closely related in structure to homeodomain proteins with linked helix-turn-helix (HTH) domains, in particular the paired domain Pax and Myb family proteins that are involved in eukaryal transcriptional regulation.

CRISPR adaptive immune systems of Archaea

“…The present work did not include an analysis of toxin–antitoxin gene pairs and IS elements that are commonly associated with archaeal CRISPR systems. 15 Nor does it cover proteins implicated in modulating CRISPR RNA transcription and processing including Cbp1 of the Sulfolobales 18 and Cbp2 of the Thermoproteales, 19 which, like RNase III that mediates RNA processing in bacterial Type II CRISPR systems, 20 are not linked genomically to CRISPR systems, and are therefore likely to perform additional cellular functions.…”

Biology and Applications of CRISPR Systems: Harnessing Nature’s Toolbox for Genome Engineering

“…Phage evasion of CRISPR immunity is another active area of research, with identified mechanisms including DNA modification, specialized anti-CRISPR proteins, and mutational escape (Bondy-Denomy et al, 2013;Bondy-Denomy et al, 2015;Bryson et al, 2015;Deveau et al, 2008;Paez-Espino et al, 2015;Pawluk et al, 2014). The context-dependent regulation of CRISPR-Cas systems in response to phage infection and stress signals has also been explored but requires further study (Bondy-Denomy and Davidson, 2014;Garrett et al, 2015;Kenchappa et al, 2013;Patterson et al, 2015;Pul et al, 2010). The rapid development of technology derived from CRISPR-Cas systems, most notably Cas9 but also Cas6f/Csy4, Cascade, and Cpf1, has fueled intense interest in the field.…”

Evolution of Homeobox Gene Clusters in Animals: The Giga-Cluster and Primary vs. Secondary Clustering

“…It is difficult to find a developmental gene network in animals that does not include a homeobox gene. These genes are taxonomically widespread, being found in animals, plants, fungi, and protists (Derelle et al, 2007;Mukherjee et al, 2009;de Mendoza et al, 2013;Mishra and Saran, 2015) and are thought to have evolved from some sort of Helix-turn-Helix protein similar to those found in prokaryotes (Laughon and Scott, 1984;Kenchappa et al, 2013). Focusing on the homeobox genes of animals, eleven classes of gene families are usually recognized: ANTP, PRD, LIM, POU, HNF, SINE, TALE, CUT, PROS, ZF, and CERS (Holland et al, 2007).…”