Scientists decided to study, in vitro, the activity of theeukaryotic DNA polymerase Pol e.
1) In a first experiment, they apply a solution of Pol e on top ofa chromatography column containing heparin. They collect thesolution that elutes from the bottom of the column (flow-through).Their analysis reveals that the flow-through does not contain anyprotein.
What is the property of heparin demonstrated by thisexperience?
2) They designed a second experiment including 4 steps.
Step 1: A long single stranded DNA (M13) was incubated with aradiolabeled primer (P) with a sequence complementary to a shortregion of M13.
Step 2: The DNA/P complex was then incubated with the DNApolymerase, the 4 types of deoxyribonucleoside 3-phosphate (dNTPs),with or without heparin.
Step 3: After incubation for different length of times (0, 1, 2,and 4 minutes) the DNA polymerase is inactivated, the DNA isextracted and separated by agarose gel electrophoresis.
Step 4: The radioactive DNA was visualized by autoradiography.
Scientists compared the activity of two DNA polymerase: andIntact polymerase (Pol e WT) and, a mutant polymerase lacking aportion of the protein (Pol e D). Not that the deletion does notaffect the catalytic activity of DNA Pol e.
The results of the experiments are listed in the figure below.
a) What does the 31 nucleotides (nt) fragment represent? Bespecific
b) Why do you think the 31 nt fragment was not detected when Pol eWT was incubated with heparin?
c) By comparing the data for Pol e WT and Pol e D and based onwhat you know about this polymerase, could you identify the domainof Pol e missing in Pol e D Briefly explain?
