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 0- N-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 EC- SOD 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.