Degree Type

Dissertation

Date of Award

2018

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

Major

Biochemistry

First Advisor

Dipali Sashital

Abstract

Prokaryotes utilize the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) – Cas (CRISPR-associated) adaptive immune system to defend against infection. A CRISPR locus consists of an AT-rich leader region followed by a series of DNA repeats interspersed by foreign DNA-derived spacers. Upon viral infection, Cas proteins acquire short fragments from the invader and insert them as new spacers into the CRISPR locus. CRISPR transcripts are generated from the CRISPR locus and assemble with Cas proteins to form the surveillance complex. The CRISPR RNA guides the complex to target foreign genetic elements bearing sequence complementarity to the crRNA and recruits a Cas nuclease for degradation. The research presented in this dissertation focuses on understanding the mechanisms of CRISPR RNA guided immunity in Bacillus halodurans type I-C system during adaptation and interference.

Cas4 is widespread across types I, II and V and is thought to be involved in spacer acquisition along with the universally conserved Cas1 and Cas2 proteins, but the role of Cas4 has remained unclear. Using a combination of biochemical and structural experiments, we reveal that type I-C Cas4 in B. halodurans interacts directly with Cas1 and Cas2, forming a Cas4-Cas1-Cas2 complex, that mediates spacer selection, processing, and integration during CRISPR immunity. Cas4 associates tightly with Cas1 and the presence of CRISPR DNA substrates helps to stabilize the higher order complex. Cas4 selectively captures spacers that contain protospacer adjacent motifs (PAMs), short sequences required for proper target recognition by the surveillance complex, and processes the substrate directly upstream of the PAM site. When in complex with Cas1-Cas2, Cas4 cleaves spacers endonucleolytically and the complex preferentially integrates the processed spacers at the leader-repeat junction in the CRISPR locus. Together, our findings demonstrate that Cas4 is indispensable in CRISPR immunity by providing functional spacers for target recognition.

For target recognition, type I-C system is unique in that only three proteins are required to form its surveillance complex. It is unknown how type I-C Cascade searches for targets using this minimal machinery. We investigated binding interactions of B. halodurans type I-C Cascade with dsDNA and found that, unlike E. coli type I-E Cascade, type I-C Cascade has much strong non-specific affinity for DNA. These observations suggest a search mechanism involving longer-lived interactions with DNA, potentially through one-dimensional sliding. To test this, we initiated development of a single-molecule fluorescence resonance energy transfer (FRET) assay to directly visualize how Cascade searches target DNA in real time. We constructed a system suitable for labeling type I-C Cascade with a fluorophore for the smFRET assay. Using this system, we detected bulk FRET between Cy3-labelled dsDNA target and Cy5-labelled Cascade upon DNA binding. These experiments established a FRET system that will be used for future smFRET experiments to understand the kinetics and mechanisms for searching DNA targets by type I-C Cascade.

Copyright Owner

Hayun Lee

Language

en

File Format

application/pdf

File Size

138 pages

Included in

Biochemistry Commons

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