DNA Polymerases

Basic Function: catalyzes Polynucleotide chain elongation
        - involves coupled endergonic/exergonic reaction:synthesis of phosphodiester bonds between nucleotide residues is coupled to the breakdown of activated substrates
 

- Synthesizes bonds in the 5' to 3' direction
 
 

                 -mechanism involves dimeric DNA polymerase
        - synthesis of leding strand occurs simultaneously with unwinding of parental       strand
        - lagging strand synthesis occurs only after a section has been unwound, discontinuously synthesizing Okazaki fragments
 
 
 
 

3 Active Sites

1.  Polymerase activity: facilitates phosphodiester bond formation
        active site lies in palm domain

2.  3' exonuclease activity: proofreading
        active site lies in base of palm

3.  5' exonuclease activity: DNA repair and RNA primer removal
 
 

Holoenzyme Assemby

-involves interactions between 3 molecules
        1. Polymerase: gp43
        2. Clamp loader: gp44/62
        3. Sliding clamp: gp45
 
 

-Clamp loader has ATPase activity that couples ATP hydrolysis to loading the sliding clamp onto DNA; acts as a chaperone for the ring-shaped clamp
- The Sliding clamp encircles the DNA and binds the polymerase to prevent frequent dissociation from the DNA, stabilizing the replication machinery
 
 

Steps
 

• clamp loader gp44/62 binds to gp45 and uses ATP hydroysis energy to open the ring
• complex binds to DNA
• gp44/62 releases the gp45 rings causing it to reclose around the DNA

Phosphodiester bond formation
 
 

T7

• structure has two magnesium ions bound to nucleutide phosphates and aspartate residues
• one metal ion polarizes the hydroxyl group at the 3' terminus, facilitating nucleophilic attack upon the alpha phosphate of the dNTP substrate
• both ions stabilize a transition state where the alpha phosphorous is linked to five oxygens
• the second ion facilitates the leaving of the pyrophosphate

 
 
 
 
 
 

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Retroviruses

   -Family of RNA viruses that possess the enzyme reverse transcriptase.
 
 

Reverse Transcriptase:

    Synthesizes a double-strand DNA molecule from a single-strand RNA template.

Method of Replication:

    *Uses viral RNA as a template for synthesizing a
      complementary DNA strand, with a specific transfer
      RNA molecule serving as a primer
 

RNA replicases:
-Enzyme that replicates the input RNA

*Lack proofreading ability

*Allow for viruses to undergo mutation and evolution very rapidly