You are studying a newly identified chromatin-remodeling complex, which you call

Question

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.            Explain the results in lanes 1–4 and why it is important to have this information before you begin to test your remodeling complex. 5 points

B.            What can you conclude about your purified remodeling complex from the results in lanes 5 and 6? 5 points

100 bp 300 bp 4 400 bp 300 bp 200 bp 100 bp _ 1. EcORI 2. DNase I 3. core octamer incubation, later treated with DNase l 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

Explanation / Answer

Lane 1 - it given a two pieces of DNA, it means the EcoRI is working and the DNA molecule has a site for EcoRI.

Lane 2- it given a degradation of DNA, so the Given DNAse I is finctional.

Lane3 - histone octamer bound the DNA, but the remaining free end of DNA (un bound to histone) is degraded with DNAse I, so a Histone bound and partial degraded DNA band was appeared.

Lane4 - Because of the Histone binding with DNA, EcoRI is unbale to cut the DNA at its site, so band is appeared of full length.

Lane 5 - Here we additionally added the NICRC and ATP, it shows the two pieces of DNA on gel, it means the NICRC mimics the DNA binding with Octamer by using ATP and so the DNA cut in two pieces and so two bands of DNA are genrated.

Lane 6 - ATP is not added, where ADP is added, so the results are the as same as lane -4. it means DNA is bound to octamer and EcoRI doen't cut it. means that the NICRC cant function in absence of ATP. it needs ATP to function.

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