Biochemical Prospective of DNA polymerase in Replication
Biologists and chemists have long recognized a relationship among DNA, RNA, and protein, and this recognition has guided a vast amount of research over the past decades and generations. The pathway of DNA to RNA and RNA to protein is conserved in all forms of life and is often called the Central Dogma. DNA functions as a storage molecule, holding genetic information for the lifetime of a cellular organism, and allowing that information to be duplicated and passed on to its progeny. Synthesis of the duplicate DNA is directed by the parental molecule and is called replication. DNA replication is an extraordinarily important complex process upon which all life depends. This process is catalyzed by DNA polymerase enzymes.
A DNA polymerase is an enzyme that catalyzes the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase reads and intact DNA strand. This process copies a piece of DNA. The newly polymerized molecule is complementary to the template strand and identical to the template’s original partner strand (Wikipedia).
In understanding the biochemical prospective of DNA polymerase in replication, its important to understand the structure of DNA and the machinery behind it. The structure of DNA provides a template-driven mechanism for its replication. Experiments by Meselson and Stahl showed that each polynucleotide strand serves as a template for a daughter strand. On completion of replication, each daughter strand, which is hydrogen bonded to its template, or parental strand, segregates to one of the daughter cells. This mode of DNA replication is called semiconservative DNA replication.
DNA polymerase requires a template, all four deoxyribonucleoside triphosphates (NTPs), and a primer from which to extend the chain, The polymerization reaction involves the nucleophilic attack of the growing DNA chain’s 3’-OH group on the alpha phosphoryl group of a free NTP that is hydrogen bonded to the template. The liberated PPi is subsequently hydrolyzed, making the polymerization irreversible. And because the 3’ end of the chain grows, polymerization is said to proceed from 5’ to 3’/DNA synthesis in vivo begins as the extension of an RNA primer synthesized by primase at the site of initiation of DNA replication. Primers are subsequently removed by the 5’ to 3’ exonuclease activity of a DNA polymerase and the polymerase fills in the gap, which is then sealed by DNA ligase. Ligation is endergonic and requires the free energy of ATP or NAD hydrolysis.
In dealing with prokaryotic DNA replication, there are three DNA polymerases. DNA polymerase I, the enzyme discovered by Arthur Kornberg, removes RNA primers via it’s 5’ to 3’ exonuclease activity and subsequently fills in the gaps via it’s 5’ to 3’ polymerase. These two reactions are referred to as a nick translation. A second polymerase of prokaryotes is the DNA polymerase II which is involved in DNA repair. And lastly, the third polymerase, DNA polymerase III, is the primary DNA replicating enzyme. It is the largest of the 3 with at least 10 subunits. Pol III has a 5’ to 3’ polymerase activity and a 3’ to 5’ exonuclease activity that eliminates misincorporated nucleotides. The polymerase active site forms sequence-independent hydrogen bonds with double-stranded DNA and thus can detect mispairings.
DNA synthesis of both leading and lagging strands is carried out by the replisome, a complex unit containing two DNA polymerase III enzymes. In order for the replisome to move a single unit in the 5’ to 3’ direction, the lagging strand template must loop around once so that the Okazaki fragment and the leading strand can be synthesixed in the same direction. The beta subunit of Pol III forms a sliding clamp that moves along DNA, allowing Pol III to replicate the DNA with a process rate greater than 5000...
Cited: Voet, Judith and Donald., and Charlotte Pratt. Fundamentals of Biochemistry: Life at the Molecular Level. Unites States of America, 2008
Wiley, Joanne., Linda Sherwood and Chrostopher J. Woolverton. Prescott, Harley, and Klein’s Microbiology. United States of America, 2008
Internet web site. www.wikipedia.com ( DNA polymerase)
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