Question

HP1 is a DNA binding protein that interacts with a specific sequence (TGCTTATTC). You want to analyze, by a Dnase I footprinting assay, the effect on DNA binding of the interaction between HP1 and its 6 binding partner PA. In each assay, you combine a radiolabeled fragment of DNA that binds to HP1 and a specific combination of proteins. After incubation with DNase I, each reaction mixture was resolved by gel electrophoresis, and then exposed to film. The autoradiogram showing the results for each combination of proteins is diagrammed below.

Lane 1: DNA alone, no protein added in the nuclease assay.

Lane 2: DNA + 5 ng (nanograms) of HP1

Lane 3: DNA + 5 ng of HP1 + 2ng of OCT1 (a protein known to interact with HP1)

Lane 4: DNA + 5 ng of HP1 + 10 ng of OCT1

Lane 5: DNA + 5 ng HP1 + 5ng PA

Lane 6: DNA + 5 ng PA

1) For each lane (1 to 6) give a brief interpretation of the results.

2) When DNA is mixed with PA and OCT1, the band pattern observed on the autoradiogram is identical to one of the pattern shown above. Which one? Explain.

3) Explain how DNA binding proteins can recognize a specific sequence without opening the DNA double helix.

Answer 1

1) lane 1: DNA alone, no protein added in the nuclease assay: Radiolabelled DNA is exposed completely to enzyme Dnase which cleaves the DNA segment and the bands are shown.

lane 2: DNA + 5 ng (nanograms) of HP1: HP1 is Dnase binding protein that binds to the specific sequence (TGCTTATTC).So that part of the DNA is prevented from enzymatic cleavage by DNase I and the portion is represented in the gel as clear area.

lane 3:DNA + 5 ng of HP1 + 2ng of OCT1 (a protein known to interact with HP1).The clear area as shown in the band shows faint visible bands which means 2ng of OCT1 interacts with 5ng of HP1 and partly hinders the binding of HP1 protein to the DNA sequence.

lane 4:DNA + 5 ng of HP1 + 10 ng of OCT1: 10ng of OCT1 interacts with HP1 protein and completely prevents the HP1 protein to bind to the DNA sequence and so the DNA is enzymatically cleaved by Dnase I enzyme.All the same bands as seen in lane 1 are seen when DNA alone is exposed to Dnase I.

lane 5:DNA + 5 ng HP1 + 5ng PA:HP1 protein binds to its specific DNA sequence.HP1 protein interacts with PA protein and together helps in binding of PA to its specific DNA sequience which might be close to the HP1 binding sequence.So, a comparatively large clear area is seen which means that the segment of DNA is protected from cleavage by Dnase I.

lane 6:DNA + 5 ng PA : PA protein does not interact with the DNA.So, DNA is exposed to cleavage by Dnase enzyme.

2.When DNA is mixed with PA and OCT1, the band pattern observed on the autoradiogram is identical to one of the pattern shown above and this one is : lane 6 and lane 4 are identical to lane 1.

Explaination: DNA is mixed with PA as in lane 6, PA protein does not bind to any sequence on the DNA and entire DNA is cleaved by Dnase.In lane 4,10ng of OCT1 completely inhibits the binding of HA protein to its specific segment on the DNA and hence entire DNA is cleaved by Dnase .So, lane 6 and lane 4 show the same band pattern as in lane 1.

• 3.The DNA binding proteins first diffuse and collide along DNA segment.The diffusion is facilitated diffusion.After that, they slide along the DNA and form a non specific interaction with a random sequence on the DNA.This is followed by intramolecular translocation to the specific site on the DNA where they bind to the specific sequence via.This process is known as sliding mechanism.
• Also, the protein might hop from one site of the DNA to the other and then bind to its specific segment.This is known as hopping mechanism.
• Another mechainsm is known as intersegmental transfer in which a protein forms an intermediate loop and associates with two DNA sites at a time as in case of Lac repressor protein.