Question 5: HP1 is a DNA binding protein that interacts with a specific sequence

Question

Question 5: 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 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.

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.

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 OCTI Lane 5: DNA +5 ng HP1 5ng PA Lane 6: DNA +5 ng PA S 6

Explanation / Answer

1) lane 1 contain no DNA binding protein and DNA is completely digested by DNase1 enzyme.

lane 2 contains DNA and HP1. HP1 binds with DNA and protects that sequence from digestion with DNase1.

lane 3 contains DNA, HP1and Oct1 protein. some of HP1 binds with DNA and protects that sequence from digestion with DNase1. some of the HP1 protein binds with Oct1 protein and thus unable to bind with DNA. the DNA which is not bound with the HP1 protein is digested by DNase1 enzyme.

lane 4 the Oct1 protein completely binds with HP1 protein and there is no HP1 protein left to bind with DNA and protect it from the DNase1 enzyme.

lane 5 contains DNA, HP1 protein and PA protein this PA protein binds with DNA-HP1 complex and further added protection of DNA from DNase1 enzyme. so a larger area of DNA remains undigested.

lane 6 contains DNA and PA protein. the result showed that PA protein alone is not capable to bind to DNA hence the DNase1 is able to digest all the DNA.

2) when more amount of Oct1 protein is added (lane 4), it is similar to the lane 1 and lane 6.

3) DNA binding proteins generally contain DNA- binding motifs that interact with DNA: this DNA- Binding domain interact with the major groove of the double helix. hence DNA does not have to be unwound for interaction.

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