Question

Little challenge from Frostbite based on present research.
Requirement: Simple molecular genetics

Answer 1

Cascade is a multifunctional nucleoprotein involved in the immune system of prokaryotes against foreign DNA. Functional cascade contains 11 subunits and a RNA stand transcribed from CRISPR loki (see attachment). It’s been analyzed that DNA that got a complementary sequence to the RNA stand is able binding to Cascade. Surprisingly DNA which does not have a complementary sequence to the RNA stand is able of binding as well somehow. I like to see if this is really true, so to a mixture we are adding the restriction enzyme (Bsml) which is able to cleavage DNA.
Data from the experiment is found in the attachment along with the figure.

A) What is a restriction enzyme?
B) What can we specifically see from the data? (What is being cleaved?)
C) What can conclude about DNA binding to Cascade from this experiment?

Data obtained from Westra et al. (2012)

Answer 2

@mrdoldum

Answer 3

@Abhisar you don't feel like giving a go your self?

Answer 4

@Abhisar as said it is a challenge so I already know the answer.

Answer 5

Ohh..i didnt noticed that..!!

Answer 6

m not much acquainted with these stuffs !

Answer 7

\(\color{blue}{\huge\ddot\smile}\)

Answer 8

nSC, OC and Lin DNA are all different DNA topology, and are not required to be known what is, to solve the problem: all you need to know is that it is not the same.

Answer 9

well as u said in the text A has the property of cleaving DNA molecules

Answer 10

B cleave is basically saying divide/ break kinda

Answer 11

That is correct yes. :)
I suggest you compare the lanes 1-4 and 5-8. and note two things:
1) Has the DNA be cut?
2) Where is the band lying

Answer 12

well for C i think reason is that bases are still same meaning A T G C

Answer 13

not, rather the chemical bond was broken

Answer 14

no*

Answer 15

between the DNA (which does not have a complementary sequence to the RNA stand) and Cascade

Answer 16

Ahhh... Lets try look at it.
In the attachment I've put some help to lane 1-4

Answer 17

hmm, well Immunity is mediated by small RNAs that guide endonucleases to cleave DNA

Answer 18

@alphadxg that is correct, but experiments on the well... "surveillance system" is capable of binding both targeting and non-targeting. The type of binding we don't know. So I test for if the bind is specific or non-specific :)

Answer 19

i have no idea how to look at it :P im not familiar with this :(

Answer 20

Is this 1 semester molecular bio?

Answer 21

@alphadxg It is simple protein science. You don't have to label stuff to different semester bio courses.

Okay. I can try give a full example. It is very stright forward. :)
A restriction enzyme is as you say @Somy an enzyme that is able cutting nucleic acids.
To make a worked example on B I look at the lane 1-4:

In 1) we only got DNA, which is going to serve as the control to this experiment. As we are not having any restriction enzyme into the solution, the DNA is uncut.

in 2) we got Cascade and DNA, we can see that the DNA still haven't been cut, as it lies next to the uncut DNA band, but still we can slightly note that the band is a little higher indicating that the DNA in fact have bounded to Cascade.

in 4) we got DNA and Bsml and the DNA most therefor have been cut. We can see that the band is much higher than the uncut DNA band and is serving as a control.

in 3) (the actual test), it got DNA, Cascade and Bsml. We can see that the band is located near the cut DNA band from lane 4).... this means that non-target DNA is being cut by Bsml.

Answer 22

You can try 5-8 if you like. Same procedure.

Answer 23

1 and 2 are same as 5 and 6

Answer 24

Exactly. So that most be that target DNA and target DNA bound to Cascade is not being cut right? :)

Answer 25

yes

Answer 26

in 7 its not located near DNA cut anymore

Answer 27

as it was in 3

Answer 28

So in the 7 it does not cut? (just need to be clear :) )

Answer 29

yes

Answer 30

in 8 the cutting happens

Answer 31

Perfect that was B! :D
So if we had to answer C what could we say there?

Answer 32

hmmm DNA is cut only if its alone with BSml?

Answer 33

i mean the targeted one

Answer 34

oh wait

Answer 35

I think you are onto the right thing :)

Answer 36

DNA that is Not complimentary is cut by BSml

Answer 37

so BSml only cuts them

Answer 38

waaaait

Answer 39

ah BSml cuts both

Answer 40

doesn't that mean the enzyme is non specific?

Answer 41

YES! exactly. To be more specific but the general idea of what you: target binds specifically to Cascade blocking the cutting of the DNA, unlike for non-target DNA, which are bound unspecific (not protected by Cascade)

Well done you analyze real data from a less than 2 year old article.

Answer 42

lol

Answer 43

that's interesting xD

Answer 44

So many people are lacking generally analysis skills, this is basically what I teach freshmen at my institute.
Ones again well done... you make your title pride! :)

Answer 45

yaaaay :D cool
thank u!!!!!!

Answer 46

I'll be posting more of these kind of questions. So if you like this kind of work, just keep an eye open :P

Answer 47

okaaay though i'll need explanation since im not really familiar with such staff
i just finished my gr 12 AS levels so i don't know much

Answer 48

Well you still did great. I mean all you in fact need to know is:
1) that a restriction enzyme cuts DNA
2) You know enzymes got a active site... well if the substrate is blocked or modified (for example a huge 11 subunit protein called Cascade place it self on it) then it can't cut. :)

Answer 49

So basically what you saw is that Cascade bind target DNA (RNA-complementary DNA) to it's active site, blocking the restriction enzyme from cutting (a specific binding).

But non-target DNA is not bound in the active site but somewhere else (perhaps on the outside of the protein) and therefor can be cut by the restriction enzyme. :)

Answer 50

in 2 u r talking about competitive and not competitive inhibition right?

Answer 51

Naaa not really, that is enzyme kinetic terms. But close.

Answer 52

non-competitive inhibition got an effect. This doesn't :)

Answer 53

so complimentary DNA is just like a normal substrate
target DNA is more like non competitive inhibitor

Answer 54

binding to a site other then active side

Answer 55

I try make a drawing a little later. :) then I try show what it means

Answer 56

okaaay thnx!! :)

Answer 57

what do u teach btw?