Biochem Genetics Helicase and Polymerase- Comp Exam Study guide.txt

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  1. Helicase mechanisms
    • 3 models of DNA unwinding:
    • Wedge model of DNA unwinding 
The hexamer at the unwiding junction interacts with one DNA strand through its central channel. The excluded strand and duplex DNA do not interact tightly. The force producing unidirectional translocation leads to separation of duplex base pairs .
    • Torsional model of DNA unwinding 
The helicase interacts tightly with both the separated strands. The rotation of one strand with respect to the other results in unwinding of duplex DNA.
    • Helix destabilizing model of DNA unwinding 
The hexamer interacs with one of the separated strands in the central channel, and also it interacts with the dsDNA on the outer parts of the hexamer. Helicases encounter a region of double helix they continue to move along their strand prying apart the double helix. The duplex region is melted by the helicase, and the helicase translocates unidirectionally along the DNA in the central channel. 

  2. Topoisomerase roles
    • Responsible for regulation of DNA pos. and neg. supercoiling
    • 1. Alter topological state of DNA
    • 2. Facilitate protein interactions w/DNA for DNA replication
    • 3. Prevent harmful excessive SUPERCOILING

  3. Helicase
    • protein that unwinds duplex DNA by HYDROLYSIS of NTP
    • Unwinding of DNA involves: unidirectional translocation and strand separation

  4. Unwinding with helicases
    • can be catalyzed by 2 DNA helicases
    • 1. T7 gp4 helicase (unidirectional translocation 5'-3' direction)
    • 2. RepA helicase
    • these move in opposite directions along ssDNA

  5. Strand separation requirements
    Requires breaking of hydrogen bonds b/w NUCLEOTIDE base pairs in dsDNA Requires presences of NTP or ATP, AND Mg
  6. Polymerase structure
    • all polymerases exhibit the following:
    • 1. 2 ALPHA helices acting as THUMB and FINGERS
    • 2. Beta sheet acts as PALM (holds DNA)
    • 3. PALM domain has 3 Asp residues that coordinate Mg ions and Catalyze polymerase reaction ◦
  7. Mechanism of POL I
    • 1. DNA rearrangement, DNA template strand moves to PALM and
    • 2. DNA held in place by conformational change in THUMB and movement of finger
    • 3. NeW dNTP moves in by binding to Mg ion (1st Mg) and FINGER region
    • 4. CLosing of fINGERS and BINDING of Aspartate
    • 5. Aspartate binds to 2nd Mg ion
    • 6. Catalysis of reaction removing pyrophosphate and ATTACHMENT to the growing DNA ◦
  8. Role of Aspartate 882
    • Serves as ANCHOR during FINGER-closing transition
    • does this by bridging the ACTIVE site - linking 3'-OH and dNTP-Mg.
    • Specifically interacts with Mg (metal B)

  9. Role of Asp705
    • 1. Enhances Catalysis, AFTER cLOSED complex forms
    • In this closed ternary complex structure, Asp rotates and binds to 1st Mg (metal B)
    • 2. Binding of ligand to 2nd Mg (metal A) after FINGER closed conformation, in prep. for Phosphoryl transfer

  10. Role of Mg ions
    • First Mg necessary for binding to dNTPs, needed in order to bind to Pol-DNA comlpex
    • 2nd Mg required for COMPLETION of catalysis. Appears to not be needed until
    • Role of Eukaryotic polymerase Alpha, delta and epsilon
    • Alpha: Involved in initiation, by making RNA primer and adding dNTPs to primer
    • Delta: can sense RNA primer and create flap for removal
    • Epsilon: pol epsilon tail may act as a built-in DNA clamp, as it mediates DNA interactions.
    • Delta and Epsilon: Needed for elongation, they replace pol alpha binding with clamps
    • Also can play a role in BLOCKING cells from entering cell cycle
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Biochem Genetics Helicase and Polymerase- Comp Exam Study guide.txt
Biochem Genetics Helicase and Polymerase- Comp Exam Study guide.txt
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