1. State what type of interaction (specific or nonspecific or both) the following
proteins might have with DNA. Speculate the regions of DNA these proteins
might be contacting.
DNA polymerase I – NS, phosphate backbone
Restriction enzymes – S, sequence of bases
TATA binding protein (TBP) – S and NS, sequence of TATA box, structure of the
minor groove in DNA
Bacterial HU protein – NS, phosphate backbone in the minor groove in DNA
Yeast transcription activator GAL4 – S, sequence of the GAL4 binding site
DNase I endonuclease – NS, phosphate backbone of DNA, some specificity for
pyrimidine bases
2.
Native gel electrophoresis can be used to separate and analyze different DNA-protein
complexes. These type of assays are often referred to as gel shift or electrophoretic
mobility shift assays. This approach can be used to measure the affinity and relative
composition of different DNA complexes. The diagram above represents a gel shift
assay of three different radiolabeled nucleosomal substrates (0-N-20, 0-N-67 and 0N-109, where N stands for nucleosome and the numbers indicate linker DNA) with
ISW2 protein complex. Lanes 1, 6 and 11 do not contain any ISW2. Competitor DNA
implies an excess amount of unlabeled exogenous DNA that competes with N for
ISW2. The gel has been exposed to phosphorimager screens that when scanned show
the positions of radiolabeled bands.
Interpret the results from the gel explaining mobility differences of bands in each
lane. How do the three substrates differ in their affinity to ISW2? – lanes 1, 6 and 11
show differences in mobility due to differences in linker DNA length. Lanes 2, 7 and
12 are fully bound by ISW2. N*ISW2 complex migrates slower than N alone.
Increasing amounts of competitor DNA displaces ISW2 from N*ISW2 complex.
Competition is most efficient in case of 0-N-20 and hence this substrate has the least
affinity for ISW2. The other two substrates have comparable affinity.
3.
An end-labelled 302bp MluI/SmaI fragment (positions -208 to +104) of the mouse ECSOD promoter was incubated with nuclear extracts obtained from mIMCD3, a mouse
kidney medulla cell line (lanes 3 and 4) and MLg, a mouse lung fibroblast cell line (lanes
5 and 6) and subsequently digested with increasing amounts of DNase I. Lane 1, G+A,
Maxam-Gilbert sequencing ladder of the probe DNA; In lane 2, the probe was incubated
with BSA.
What kind of assay is represented in the illustration? – DnaseI footprinting
Elaborate on the assay you named above – see class notes
What does A-E indicate? – binding sites for nuclear proteins from the mIMCD3 extracts
on the EC-SOD promoter
Interpret the results shown in the illustration – proteins from the mIMCD3 extracts bind
to EC-SOD promoter fragment at 5 sites but those from MLg do not bind
How do you think the results are considered to be specific? – lane 2, specificity control
with BSA. BSA does not to the EC-SOD promoter.