You Are Studying a Newly Identified Chromatin-Remodeling Complex, Which You

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 5-67A 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 5-67B 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)

(B)

1. ECORI
2. DNase I
3. core octamer incubation, later treated with DNase I
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 5-67
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?