NUCLEIC ACID DEGRADATION BY THE CRISPR TYPE III-B COMPLEX

CRISPR-Cas;RNA;DNA;Nuclease;CMR;Biochemistry

Estrella, Michael AnthonyPrigge, Sean T. ;

Johns Hopkins University

关键词: CRISPR-Cas;    RNA;    DNA;    Nuclease;    CMR;    Biochemistry;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/39742/ESTRELLA-DISSERTATION-2016.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system is an adaptive immune system utilized by prokaryotes to preserve their genome against invasive nucleic acid. CRISPR-derived RNA (crRNA) and CRISPR associated (Cas) proteins form an effector complex that degrades foreign genetic elements and is the basis of this prokaryotic defense system. The CRISPR Type III-B effector complex, also referred to as the Cmr complex, is composed of 6 protein subunits (Cmr1-6) and a crRNA. It has been shown to cleave ssRNA in vitro and degrade transcriptionally active DNA in vivo. To understand the mechanism of this activity, we undertook a biochemical analysis of the Cmr complex to determine how it cleaves nucleic acid.We have been able to recombinantly express and purify the individual Cmr subunits of Thermotoga maritima MSB8, to form the Cmr complex with a short crRNA in vitro. Using pull-down assays we demonstrate that Cmr proteins from T. maritima assemble into a Cmr complex with the same subunit interactions as those observed for Cmr complexes from other species. Furthermore, we have reconstituted CRISPR-mediated degradation of RNA in vitro. Analysis of this activity demonstrates the preference for cleavage of ssRNA at 6 nucleotide intervals and the dependence on the 2’ hydroxyl adjacent to the scissile phosphate for cleavage. We also report ssDNA cleavage activity by the Cmr complex, which was previously unknown. Cleavage is observed only at thymidine nucleotides and is dependent upon the presence of target RNA complementary to the crRNA of the complex. We also demonstrate that as complementary target RNA is degraded, DNA activity is turned off. We mapped the active site for ssDNA cleavage to the HD domain of the Cmr2 subunit.Our in vitro results are consistent with transcription-coupled DNA targeting by the Cmr complex. This study further resolves the ambiguity in activities reported for Type III systems and supports a unified mechanism for all Type III systems in which DNA nuclease activity is coupled to transcription.