Question

8. You are studying a newly identified chromatin-remodeling complex, which you call NICRC. You decide to run an in vitro experiment to characterize the activity of the purified complex. Your molecular toolbox includes: (1) a 400-base-pair DNA molecule that has a single recognition site for the restriction endonuclease EcoRI, an enzyme that cleaves internal sites on double-stranded DNA (dsDNA); (2) purified EcoRI enzyme; (3) purified DNase I, a DNA endonuclease that will cleave dsDNA at nonspecific sites if they are exposed; and (4) core octamer histones. You are able to assemble core nucleosomes on this DNA template and test for NICRC activity. Figure Q5-60A illustrates the DNA template used and indicates both the location of the EcoRI cleavage site and the size of the DNA fragments that are produced when it cuts. Figure Q5-60B illustrates how the DNA molecules in your experiment looked after separation according to size by using gel electrophoresis. Your experiment had a total of six samples, each of which was treated according to the legend below the gel. The sizes of the DNA fragments observed are indicated on the left side of the gel. (A) 100 bp 300 bp (B) 1 2 3 4 5 6 400 bp- 300 bp- 1 1 1 200 bp 100 bp - 1 1. EcoRI 2. DNase 3. core octamer incubation, later treated with DNase! 4. core octamer incubation, later treated with EcoRI 5. core octamer incubation, addition of NICRC + ATP, later treated with EcoRI 6. core octamer incubation, addition of NICRC + ADP, later treated with EcoRI Figure Q5-60 A. Explain the results in lanes 1-4 and why it is important to have this information before you begin to test your remodeling complex. B. What can you conclude about your purified remodeling complex from the results in lanes 5 and 6?

Answer 1

answer A.

lane 1 : gives you the digested band of your DNA fragment by ECOR I. it shows two bands - one for 100 bp and of 400 bp which are the byproduct of digestion

lane 2: gives you the digested band of your DNA fragment by DNaseI. it shows SPREADED band which shows the DNA fragment was fully exposed to be cut by the DNaseI at every possible position giving variable size of DNA fragments.

lane 3: it shows that DNA fragment was incubated with octamer which made a DNA octamer complex. now here DNA frament was not free to be digested by DNase I and thats why we got one single band.

lane 4: here again DNA fragment was incubated with octamer which made a DNA octamer complex, after this it was incubated by ECORI. but again internal site of DNA was not exposed and that is why there is no digested bands.

reason of need of this above experiments: these types of experiments are called CONTROL experiment. the results obtained will act as control and further results will going to be compared with these to fall on some definite conclusion.

answer B:
lane 5 and 6 results have shown that once you provide NICRC and ATP to ECORI then only it can digest DNA from octamer complex which means that for exposing the DNA form octamer complex there is need of energy in form of ATP. NICRC will only act by utilising ATP molecules.