Release date: 2017-09-08
Professor Ailong Ke
On September 4th, the research team's latest achievement "How type II CRISPR–Cas establish immunity through Cas1–Cas2-mediated spacer integration" was published in Nature, and proposed a mechanism framework for the stepwise interval integration of the Type II CRISPR system. This will help to increase understanding of the mechanism of action of the Type II CRISPR system.
Bacteria face constant attacks from viruses and invading nucleic acid strand plasmids. In order to survive such an attack, bacteria and archaea use a variety of defense mechanisms, including a DNA element centered on the clustered regular interspaced short palindromic repeat (CRISPR). Adaptive immune system. The CRISPR DNA element is composed of a number of "repetitive sequence" components of 30-60 nucleotides in length that are separated by a "spacer" component that varies in length from 30 to 60 nucleotides.
To remember the infection, the CRISPR system grabbed a short sequence from the invading viral DNA and inserted it directly into the bacterial genome. Some phage DNA fragments are stored in specific regions of the genome; these form immune memory. In subsequent infections, CRISPR used these sequences to construct short RNA strands that match the phage genetic sequence. The protein complex that binds to this RNA then identifies the phage DNA and then destroys it.
If you mistakenly grab a fragment of your own DNA, it can cause bacterial cells to develop an autoimmune disease that attacks its own DNA, which can be fatal to bacteria. So how does the CRISPR system know to insert foreign DNA rather than its own DNA fragments into immune memory?
Previous studies have shown that bacteria have two proteins, called Cas1 and Cas2, which are part of the CRISPR system and are responsible for obtaining foreign DNA fragments. The CRISPR system successfully integrates plasmid DNA into the genome of bacteria, while "self" DNA is rarely attacked. The conserved Cas1 and Cas2 proteins form an integrase complex: a Cas1 dimer on the side and a Cas2 dimer in the middle.
For the E. coli IE-type CRISPR system that has been studied more, the interval sequence depends on the bacterial Integration Host Factor (IHF), but for the II-A CRISPR system, this is still an unsolved mystery. In this article, the researchers analyzed the key structure of the four structures of Cas1-Cas2 during interval integration by analyzing the Streptococcus II-A CRISPR system.
The researchers found that there are three important molecular events in the target target: first, Cas1-Cas2/prepace randomly searches for a semi-binding site and then preferentially interacts with the leading-edge CRISPR repeat, catalyzing a nucleophilic attack, ie, the leader - The proximal repeat is ligated to the 3'-overhang of the prepacer. In addition, the researchers pointed out that the recognition of the semi-binding site of the spacer sequence requires DNA bending and complete integration.
This study proposes a framework for the step-by-step sequence integration of the Type II CRISPR system, which will help to increase understanding of the mechanism of action of the Type II CRISPR system.
In July of this year, Professor Ke Ailong's research group published the near-atom resolution structure of the type I CRISPR complex of Thermobifida fusca, and revealed the mechanism of action of CRISPR-Cas3 system.
Using biochemical techniques and cryo-electron microscopy, the researchers obtained stable complex structures under different functional states, and observed that the CRISPR complex targets the target DNA strand and prepares to cleave DNA through the Cas3 enzyme. The researchers say that these structures help reveal the process of multi-layer error detection, which prevents accidental genomic damage.
The researchers also found that in CRISPR-Cas3, crRNA was added to the CRISPR protein complex. When the CRISPR complex found the PAM sequence, it would bend the DNA at an acute angle, forcing a small piece of DNA to melt. This allows a long 11 nt fragment of the crRNA to bind to the target DNA strand, forming a "seed bubble." This structure acts as a fail-safe device to check whether the target DNA matches the crRNA, and if they match correctly, the bubbles expand and the remaining crRNA binds to its corresponding target DNA to form a so-called "R-loop" structure.
Once the R loop is fully formed, the CRISPR complex undergoes a conformational change that locks the target DNA. It also allows the second non-target strand of DNA to form a bulge, thus forming a complete R-loop structure that can be cleaved by the Cas3 enzyme.
Original search
"How type II CRISPR–Cas establish immunity through Cas1–Cas2-mediated spacer integrationâ€
Source: Biopass
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