Biochem Genetics Replisome and Telomerase- Comp Exam Study guide.txt

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Biochem Genetics Replisome and Telomerase- Comp Exam Study guide.txt
2014-11-18 18:58:09
Biochem Genetics Replisome Telomerase Comp Exam Study guide

Biochem Genetics Replisome and Telomerase- Comp Exam Study guide.txt
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  1. ◦ Replisome structure components
    • DNA helicase: unwinds DNA
    • Primase: makes short RNA primers to initiate DNA synthesis
    • DNA Polymerases (III): involved in leading and lagging strand synthesis
    • ssDNA binding proteins: lagging strand protects ssDNA from nucleases and inhibits 2ndary structure
    • Circular sliding clamps and clamp loading complex: proteins encircle duplex and tether polymerase to DNA for high processivity and replication rate
  2. Clamp loader
    • Ring shaped protein structures: Bind to DNA strands and aid in DNA polymerase binding
    • Aids in regulation of initiation of replication
    • INCREASES RATE of replication                   
    • Interacts with DNA proteins involved in DNA REPAIR
  3. Clamp loading mechanism
    • Once clamp is opened conformation (via ATP binding to trigger arrangement of AAA+ ATPases)compared to its closed circle …
    • 1. the Primer Template DNA threads through the gaps: interacting with hydrophobic pocket of C-terrminus tail
    • 2. DNA Pol III acts: Displaces the ssDNA at this hydrophobic pocket
    • 3. duplex DNA (dsDNA): located inside clamp loader
    • Clamp is at an angle, possibly to allow for polymerase switching to occur
    • In order for clamp to get around dsDNA…
    • 1.ATP BINDING: occurs to open the clamp with clamp loader in place and allow binding to DNA phosphate backbone
    • 2. ATP HYDROLYSIS at B subunit, breaks interface at AAA+ of B & C: allows CLOSURE of CLAMP of clamp around primer-template DNA
    • 3. ATP HYDROLYSIS of C & D, dissolve AAA+ modules: clamp LOADER EJECTED & clamp left loaded onto DNA
  4. Lagging strand trombone cycle
    • Mediated by CLAMP loader and sliding clamps
    • 1. lagging strand DNA loop made via Pol III core-B as it extends Okazaki fragment and through fork progression producing ssDNA.
    • 2.DnaG primase synthesizes new RNA primer
    • 3. clamp loader loads new Beta clamp on RNA primer
    • 4. when Lagging strand synth. complete, lagging Pol III core disengages from its clamp
    • 5. this Pol III now associates w/NEW beta clamp (via clamp loader) at upstream primer to start new Okazaki synthesis
    • NOTE: Clamps accumulate after Okazaki fragments occur before being unloaded, and this may also serve as a marker for newly synthesized DNA
  5. Telomerase - Components of catalytic core
    • Made up of:
    • 1. Integral Telomerase RNA (TR)
    • 2. Telomerase Reverse Transcriptase (TERT)
    • These 2 components needed for TELOMERASE ACTIVITY
  6. Integral Telomerase RNA (TR)
    • This is NON-coding RNA
    • Provides template for telomere repeat sequences and motifs
    • Roles:
    • 1. binding catalytic TERT protein and various telomerase acessory proteins
    • 2. Defining template boundary
  7. TR structure
    • 3 main domains
    • 1.template pseudoknot domain: defines template boundary
    • 2.CR 4/5 domain: aids in TERT binding and telomerase function                              
    • 3.H/ACA domain: binding site for proteins
  8. Telomerase Reverse Transcriptase (TERT)
    • protein and catalytic component of core telomerase enzyme
    • main fxn: nucleotides add'n/elongation of telomere ends
  9. TERT structure
    • 4 domains
    • 1. Telomerase essential N-terminal domain
    • 2. Telomerase RNA binding domain
    • 3. Reverse transcriptase domain
    • 4. C-terminal extension (CTE), aids DNA binding by associating w/phosphodiester backbone of DNA/RNA hybrid
  10. Structure and function of TERT
    • CTE (Thumb): aids DNA binding by associating w/phosphodiester backbone of DNA/RNA hybrid
    • RNA-interacting domain 2: binding site for TR
    • CR 4/5 domain Motifs (Fingers): finger motifs 1 & 2, bind incoming nucleotides palm
    • motif A & E (Palms): form catalytic site