In DNA polymerases, the 3’ hydroxyl end attacks the phosphorus on the incoming n
ID: 3166378 • Letter: I
Question
In DNA polymerases, the 3’ hydroxyl end attacks the phosphorus on the incoming nucleotide triphosphate to add another nucleotide to the growing chain. a) These enzymes require Mg2+. What function might this cofactor serve in the enzymatic process? b) How does the mechanism explain the inability of the polymerase to synthesize DNA using nucleotide monophosphates? c) How does the mechanism explain how some DNA polymerases can replace RNA primers and damaged DNA (“nick translation,”) but not seal the backbone (“ligation”)?
Explanation / Answer
a. Mg 2+ is required for following reasons:
- During replication DNTp's gets broken down to DNMp's to form phosphodiester bond between 3' OH of adjacent nucleotide and 5' Phosphate of the upcoming nucleotide. in this reaction Mg++ binds to the alpha phosphate group of dntp and helps in the removal of beta and gamma Phosphate from dntp.
- The 3'-OH of the growing chain contains a lone pair of electron which is used for the formation of phosphodiester bond during the process of polymerization by Taq Polymerase (or any polymerase). This lone pair of electrons can do nucleophilic attack on the phosphate atom of alpha-phosphate, releasing the beta and gamma phosphate. But the incoming nucleotide has 4 negative charges (dNTP4-) and due to the presence of these many negative charges the nucleophilic attack is retarded. Now, Mg2+ comes to rescue by chelating extra negative charges of the incoming dNTP, facilitating the nucleophilic attack and bond formation, hence, polymerization.
- It is also proved that MG2+ binding with nucleotide is also required for closing the active site befor chemical reaction which help guide polymerase selection for the correct nucleotide extention.
b. Ans:
DNA polymerases are the enzymes that replicate DNA in living cells. They do this by adding individual nucleotides to the 3-prime hydroxl group of a strand of DNA. The process uses a complementary, single strand of DNA as a template.The energy required to drive the reaction comes from cutting high energy phosphate bonds on the nucleotide-triphosphate's used as the source of the nucleotides needed in the reaction.
DNA polymerases can not create new strands of DNA. They only synthesis double stranded DNA from single stranded DNA. The starting point is a a stretch of single stranded DNA which is double stranded for at least part of its length. In the polymerase chain reaction the double stranded stretch is created by attaching short DNA primers. In living cells, RNA primers are used. DNA polymerase uses the bases of the longer strand as a template. During strand elongation, two phosphates are cleaved from the incoming nucleotide triphosphate and the resulting nucleotide monophosphate is added to the DNA strand. This results in the:
Nucleotide monophosphate will not able to provide the energy required for extension and adittion of nucleotide at 3' end.
c. DNA polymerase can't start working without 3'OH extremity and template to copy. For replication, primase is synthesizing short RNA primer to generate 3'OH extremity. On the leading strand a unique RNA primer is required as DNA synthesis goes the same direction as the replication fork, on the lagging strand the synthesis goes opposite way to fork progression but the full replication machinery (which is actually working on both strands at the same time) has to follow the fork progression which is why replication is discontinuous : primase is generating RNA primer, DNApolIII is filling the gap between two RNA primers and then DNApolI is replacing RNA primer by DNA. DNA polymerase III requires template to perform its activity and when there is nick DNA polymerase III is not having any template or it does not required addition of nucleotide. Ligase is the enzyme which forms the phophodiester bond between nick created by removal of DNA damage or RNA primers.