User:Humanpersonfromhere2/sandbox

My work on Wikipedia
My main focus was to add information about how CRISPR integrates new spacers in E. Coli. The CRISPR page had good information about the Ca1-Cas2 complex and its responsibility in integration new spacers. They did not have any mention that in E. Coli IHF is responsible for the chronological addition of new spacers so I added that. I then searched to see if there was any page on IHF. What I found was a page for bacterial DNA binding proteins. On this page there was a mention of what IHF is, but had no information on its role in some CRISPR systems. I added a brief addition describing how it is used in CRISPR, and slightly re organized the section so it made more sense. Unfortunately I wasn't able to add too much more as there is still a lot unknown about how this process is carried out, this is partially why my proposal is very relevant!

Spacer acquisition
When a microbe is invaded by a virus, the first stage of the immune response is to capture viral DNA and insert it into a CRISPR locus in the form of a spacer. Cas1 and Cas2 are found in all three types of CRISPR-Cas immune systems, which indicates that they are involved in spacer acquisition. Mutation studies confirmed this hypothesis, showing that removal of cas1 or cas2 stopped spacer acquisition, without affecting CRISPR immune response.

Multiple Cas1 proteins have been characterised and their structures resolved. Cas1 proteins have diverse amino acid sequences. However, their crystal structures are similar and all purified Cas1 proteins are metal-dependent nucleases/integrases that bind to DNA in a sequence-independent manner. Representative Cas2 proteins have been characterised and possess either (single strand) ssRNA- or (double strand) dsDNA- specific endoribonuclease activity.

In the I-E system of E. coli Cas1 and Cas2 form a complex where a Cas2 dimer bridges two Cas1 dimers. In this complex Cas2 performs a non-enzymatic scaffolding role, binding double-stranded fragments of invading DNA, while Cas1 binds the single-stranded flanks of the DNA and catalyses their integration into CRISPR arrays. A key part of this system is that new spacers are always added at the begining of the CRISPR next to the leader sequence. In E. Coli a histone like protein called integration host factor (IHF), which binds to the leader sequence, is responsible for the accuracy of this integration.

IHF
Integration host factor, IHF, is a nucleoid-associated protein only found in gram negative bacteria. It is a 20 kDa heterodimer, composed of α and β subunits that bind to the sequence 5' - WATCAANNNNTTR - 3' and bends the DNA approximately 160 degrees. The β arms of IHF have Proline residues that help stabilize the DNA kinks. These kinks can help compact DNA and allow for supercoiling. The mode of binding to DNA depends on environmental factors, such as the concentration of ions present. With a high concentration of KCl, there is weak DNA bending. It has been found that sharper DNA bending occurs when the concentration of KCl is less than 100 mM, and IHF is not concentrated.

IHF was discovered as a necessary co-factor for recombination of λ phage in to E.coli. In 2016 it was discovered that IHF also plays a key role in CRISPR type I and type II systems. It has a major role in allowing the Cas1-Cas2 complex to integrate new spacers into the CRISPR sequence. The bending of the DNA by IHF is thought to alter spacing in the DNA major and minor grooves, allowing the Cas1-Cas2 complex to make contact with the DNA bases. This is a key function in the CRISPR system as it ensures that new spacers area always added at the beginning of the CRISPE sequence next to the leader sequence. This directing of integration by IHF ensures that spacers are added chronologically, allowing better protection against the most recent viral infection.