The Large C-Terminal Domain Of The Klenow Fragment Case Study

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Q1) What secondary structures make up the large C-terminal domain of the Klenow fragment?
Answer: The large C-terminal domain contains a six-stranded antiparallel β-sheet at the bottom of the cleft region and contains three α-helices on one side and four α-helices on the other side of the cleft region.

Q2) Describe the three-dimensional structure of the large C-terminal domain of the Klenow fragment?
Answer: The large C-terminal domain contains a significantly deep cleft, with a considerable area to enable the binding of the B-DNA. The overall domain itself is shaped almost like a cupped right hand where, in this case, α-helices on either side of the cleft form the thumb and fingers, while the β sheet forms the palm of the hand.

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Q4) What are the two domains contained in the Klenow fragment and what are their functions in DNA Replication?
Answer: The C-terminal fragment of DNA polymerase I consists of two out of three active domains, together known as the Klenow fragment. One of the domains acts as the binding site for DNA during DNA replication and performs the polymerase activity, adding nucleotides to the growing newly synthesized DNA chain using the existing DNA as template. The other domain consists of 3’ to 5’ editing exonuclease activity which repairs incorrectly base paired nucleotides added to the newly synthesized chain, thereby enhancing the fidelity of DNA synthesis.

Q5) How many amino acids are in each of the domains of the Klenow Fragment as well as the total amino
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Answer: Yes. The amino terminus of the helix H1 fits perpendicularly into the minor groove of the double stranded DNA.

Q8) What amino acid residues in the enzymes catalytic site would be expected to interact with the DNA? Why?
Answer: The phosphate group of the DNA backbone that the enzyme interacts with contains several negatively charged oxygen groups. Therefore, it makes sense that positively charged residues such as the R group of lysine would be expected to interact with the oxygen groups of phosphate. Furthermore, these oxygen groups can form strong and stable hydrogen bonds and thus, they would be likely to form hydrogen bonds with the R groups of amino acids such as asparagine.

Q9) Give examples of amino acids residues found in the catalytic site of the DNA polymerase that interact with DNA?
Answers: Some of the positively charged amino acids that interact with DNA include Lys-635, while examples of amino acids that can hydrogen bond with the oxygen in the phosphate backbone of the DNA include Asn-675 and Asn-678.

Q10) What structural properties of the large domain make it suitable to bind

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