As a researcher investigating structure-function questions about DNA polymerase,

Question

As a researcher investigating structure-function questions about DNA polymerase, you are interested in isolating E. coli mutants with altered DNA polymerase function and then identifying the amino acids altered in the mutants. Based on our current understanding of DNA polymerase function, predict what kind of amino acid/gross structural changes in DNA polymerase will cause the following mutant phenotypes you observe in your cells: the mutant DNA polymerase: is able to incorporate rNTPs and dNTPs into new strand loses all projectivity - shows only one reaction per binding has trouble "catching" new dNTPs - they touch down but often detach again is much more error prone than normal DNA polymerase can't form a new phosphodiester bond

Explanation / Answer

There are 3 types of DNA polymerases in E.coli DNA - Polymerase I , DNA poymerase II, DNA polymerase III DNA pol I - Removes RNA primer, DNA repair, Filling up of missing nucleitides. DNA pol II - DNA repair DNA Pol III - Replication, Proof reading and editing. Active site mutation in the DNA polymerase causes error prone addition of nucleotides. Tyr - 766 residue is important residue which is most needed for the proper function of DNA polymerase I in E.coli. Tyr - 530 in e.coli DNA polymerase I forms hydrogen bonds with Glu - 480 . This forms a binding pocket for incoming nucleotides and are able to differentiate rNTPs. When Tyr is substituted for Ser , the activity of enzyme polymerase doesnot change much but the affinity for dNTPs decreases thus detaching occurs. Thus this part may be mutated in above research which decreases the catalytic efficiency and fideility of the polymerase. This mutation also makes the DNA pol unable to differentiate rNTPs and dNTPs. Thus , it is error prone due to mismatch and unable toi bind nucleotides.

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