Biochemical Study of CRISPR-associated and anti-CRISPR proteins

630;

농업생명과학대학 농생명공학부 ;

University:서울대학교 대학원

关键词: 630;   

Others  :  http://s-space.snu.ac.kr/bitstream/10371/143670/1/000000152130.pdf

美国|英语

来源: Seoul National University Open Repository

【 摘 要 】

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR associated (Cas) proteins provide an adaptive immune system of bacteria and archaea against invading foreign nucleic acids.In type I-F CRISPR-Cas system, Cas proteins (Csy1-4) form a surveillance complex with CRISPR RNA (crRNA) to recognize the target nucleic acids. In this study, I report the biochemical characterization of Csy1-Csy2 heterodimer from Xanthomonas albilineans (XaCsy1-Csy2 heterodimer) by analyzing its interaction with crRNA and AcrF2, an anti-CRISPR (Acr) protein from a phage infecting Pseudomonas aeruginosa. Electrophoretic mobility shift assays revealed that the binding of XaCsy1-Csy2 heterodimer to the 5ʹ-handle of the crRNA is sequence-specific. Size exclusion chromatography and isothermal titration calorimetry analyses demonstrated tight binding between the AcrF2 and XaCsy1-Csy2 heterodimer. Furthermore, the X-ray crystal structure of AcrF2 was solved to a resolution 1.34 Å and enabled a more detailed structural analysis of the residues involved in the interactions with the Csy1-Csy2 heterodimer. These results provide biochemical information of the Csy1-Csy2 heterodimer from a previously uncharacterized bacterial species and also suggest that the AcrF2 protein has broad specificity in inhibiting the type I-F CRISPR-Cas system.