Dr Hifzur R Siddique Section of Genetics Department of Zoology ALIGARH MUSLIM UNIVERSITY Significance of Antiparallel arrangement role of primer exonuclease activity of DNA pol III DNA Polymerases Use a Single Active Site to Catalyze DNA Synthesis ID: 1012028
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1. Class IXM.Sc.-Semester IIDr. Hifzur R SiddiqueSection of GeneticsDepartment of ZoologyALIGARH MUSLIM UNIVERSITYSignificance of Antiparallel arrangement, role of primer, exonuclease activity of DNA pol III
2. DNA Polymerases Use a Single Active Site to Catalyze DNA SynthesisDNA polymerase uses a single active site to catalyze the addition of any of the four dNTPs. DNA polymerases show an impressive ability to distinguish between rNTPs (10X) and dNTPs (incorporation rate 1000X).DNA polymerase accomplishes this catalytic flexibility by exploiting the nearly identical geometry of the A:T and G:C base pairs.
3. Incorrect alignment reduces the rate of catalysis dramatically, resulting in the DNA polymerase preferentially adding correctly base paired dNTPs.
4. THE STERIC CONSTRAINTS PREVENTING DNA POLYMERASEFROM USING rNTP PRECURSORS. Binding of a correctly base-paired dNTP to the DNA polymerase. Under these conditions, the 3`-OH of the primer and the a-phosphate of the dNTP are in close proximity.In DNA polymerase, the nucleotide-binding pocket cannot accommodate a 2`-OH on the in-coming nucleotide.
5. This space is occupied by two amino acids that make van der Waals contacts with the sugar ring. Changing these amino acids to other amino acids with smaller side chains (e.g., by changing a Glu to an Ala) results in a DNA polymerase with significantly reduced discrimination between dNTPs and rNTPs. In this state, the a-phosphate is incorrectly aligned with the 3`-OH of the primer, dramatically reducing the rate of catalysis.
6. DNA Polymerases Resemble a Hand That Grips the Primer: Template JunctionThe recently synthesized DNA is associated with the palm, and the site of DNA catalysis is located in the crevice between the fingers and the thumb. The single-stranded region of the template strand is bent sharply and does not pass between the thumb and the fingers.
7. The palm domain is composed of a beta sheet and contains the primary elements of the catalytic site. In particular, this region of DNA polymerase binds two divalent metal ions (typically Mg2+ or Zn2+) that alter the chemical environment around the correctly base-paired dNTP and the 3`-OH of the primer.
8. The two metal ions (shown in green) are held in place by interactions with two highly conserved ASPARTATE residues.One metal ion reduces the affinity of the 3`-OH for its hydrogen. This generates a 3`O- that is primed for the nucleophilic attack of the a-phosphate of the incoming dNTP. The second metal ion coordinates the negative charges of the b-phosphate and g-phosphate of the dNTP and stabilizes the pyrophosphate produced by joining the primer and the incoming nucleotide.Two metal ions bound to DNA polymerase catalyze nucleotide addition.
9. What are the roles of the fingers and the thumb?The fingers are also important for catalysis. Several residues located within the fingers bind to the incoming dNTP. Once a correct base pair is formed between the incoming dNTP and the template, the finger domain moves to enclose the dNTP .This closed form of the polymerase “hand” stimulates catalysis by moving the incoming nucleotide into close contact with the catalytic metal ions.
10. The thumb domain is not intimately involved in catalysis. Instead, the thumb interacts with the DNA that has been most recently synthesized.This serves two purposes.First, it maintains the correct position of the primer and the active site. Second, the thumb helps to maintain a strong association between the DNA polymerase and its substrate. This association contributes to the ability of the DNA polymerase to add many dNTPs each time it binds a primer: template junction.
11. DNA Polymerases Are Processive EnzymesRapid, add as many as 1000 nucleotides/sec to a primer.Speed of DNA synthesis is largely due to the processive nature of DNA polymerase.PROCESSIVITY is a characteristic of enzymes that operate on polymeric substrates.In the case of DNA polymerases, the degree of processivity is defined as the average number of nucleotides added each time the enzyme binds a primer : template junction.
12. Each DNA polymerase has a characteristic processivity that can range from only a few nucleotides to more than 50,000 bases added per binding eventIn a typical DNA pol reaction, it takes 1 sec for the DNA pol to locate and bind a primer: template junction. Once bound, addition of a nucleotide is very fast (in the millisecond range). Thus, a completely non-processive DNA polymerase would add 1 bp/sec. In contrast, the fastest DNA polymerases add as many as 1000 nucleotides/sec.
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