Lecture 4: DNA Replication and Telomere Maintenance

1. Semiconservative model of DNA replication (ON TEST)

Three possible modes of replication hypothesized based on Watson and Crick’s model:

• Semiconservative (one strand transfered to next gen)

• Conservative (uses the model to make another dna)

• Dispersive

  • Molecuecule broken into pieces and then randoomely inserted into 2 molecules (intertwined)
    

The Meselson-Stahl experiment demonstrated that DNA replication is semiconservative

14N is most abundant form of N

• grow ecoli with 14N, as all DNa made of 14N

• exp

  • grow ecoli in the highly isotope N15 media then transfered to 14N media and allow to grow. The add CsCL(density 1.7) then centrifuge to get seperation between light(14N, top) and heavy(15N, bottom) dna

  • CsCl is used as a resolution to mix them

Meselson experiment for semi/comservative (exam)

• know band for each and ratio

DNA Replication in Prokaryotes

DNA polymerases are the enzymes that catalyze DNA synthesis from 5′ to 3′

• ONLY add nucleotides in 5′ → 3′

• CANNOT initiate DNA synthesis de novo.

  • REQUIRES a primer with free 3’-OH group at end

• Deoxynucleoside 5′ triphosphates (dNTPs) added one at a time to the 3′ hydroxyl end of the DNA chain

• dNTP add determined by complementary base paring

• As phosphodiester bonds form, the two terminal phosphates are lost, making the reaction essentially irreversible.

  
  

Leading vs Lagging strand

• Leading

  • made continuously in same direction as replication fork’

• Lagging Strand

  • made semi-coontinuously in opp direction as replication form.

  • DNA made in short segments called Okazaki fragments

→ Nucleotides added to both strands at same time and rate by 2 DNA polymerase

Replication is mediated by replisome

• Helicase

  • unwinds the parental double helix

• 2 molecules of DNA polymerase 3

• Primase (RNA Primer- transcription)

  • initiates lagging strand Okazaki fragments

• 2 sliding clamps

  • attach DNA polymerase to DNA

• clamp loader

  • use ATP to open/close sliding clamps

• SSB (single-strand DNA binding proteins

  • protect DNA from nuclease attack

  • stabalise single strand DNA template

• Topoisomerase

  • release strains caused by upwinding/winding DNA

Multi-protein machines mediate bacterial DNA replication

Bacterial DNA polymerases have multiple functions

• DNA polymerase I

  • 5′→3′ Polymerase to synthesize DNA (very short)

  • 3′→5′ exonuclease(remove nucleotide) to backtrack (proofreading)

  • 5′→3′ exonuclease to remove primer (DNA repair)

• DNA polymerase III

  • Main replicative polymerase, more processive.

  • 5′→3′ Polymerase to synthesize(ADD) DNA (very short)

  • 3′→5′ exonuclease to backtrack (proofreading)

Initiation of replication

• origin

  • where bidirectional replication fork initiates

• some bacteria have a single, well-defined origin

Topoisomerases relax supercoiled DNA

• Positive supercoiling ahead of the fork and negative supercoiling in the wake of the fork

• DNA topoisomerase

  • helps relieve torsinal strain that inhibits fork movements

Lagging strand synthesis by the replisome

• As replication fork advances the lagging strand polymerase stays with a loop

• DNA polymerase 1 remove RNA primers and replace them with complementary dNTPs

• DNA ligase catalyzes formation of phosphodiester bond btw adjacent Okazaki fragments.

3. DNA replication in Eukaryotes

   in nucleas, dna organised in nucleosome, and it has linear dna, and need to remve histones, dna is large and needs multiple origins

Potential issues:
1: Nucleosome
2: Linear DNA
3: Multiple origins

Prereplication complex formation and replication licensing

• Replication restricted to S phase of cell cycle

• Origin selection sep from initiation

• prevents overreplication of the genome

RNA priming of leading and lagging strand DNA synthesis

DNA polymerase (pol) “alpha” and its associated primase activity

• synthesizes RNA primers in eukaryotes

Polymerase Switching

Def- hand-off of DNA template from one polymerase to another

• Leading strand: switch from DNA polymerase “alpha” to pol “epsilon”
  * Lagging strand: switch from pol “alpha” to pol “Delta”

Proofreading

• Replicative polymerases are high fidelity but not perfect: 10-4 to 10-5 errors per base pair.

• Proofreading using 3' to 5' exonuclease activity reduces the error rate to 10-7 to 10-8 errors per base pair

Removal of primers

• RNA primer removed by FEN-1 and/or Rnase H

Topoisomerase untangles the newly synthesized DNA

• in eukaryotes: rep continues until 1 form meets adjacent replicon fork

• progency DNA molecules remain intertwined

• Topoisomerase 2 req to resolve the two separate progeny genomes

4. Telomere maintenance

• The end replication problem

  • When final primer removed, a 8-12 nucleotide region is left Un-replicated. which predicts that chromosomes get shorter with each replication round

Telomers

• Eukaryote chromosomes end with tandem repeats of a simple G-rish sequence

  • Humans: TTAGGGG

  • Tetrahymena: TTGGGG

• seal ends of chromosomes

• creates stability by keeping chromosomes from ligating together

Carol Greider and Elizabeth Blackburn discovered Telomere

• studied etrahymena thermophila, a single-celled eukaryote with over 40,000 telomeres

• DISCOVERED the enzyme TELOMERASE

Maintenance of telomeres by telomerase

• Telomerase elongates the 3′ end of the template for the
  lagging strand (G-rich overhang).

• Telomerase is a ribonucleoprotein (RNP) complex with
  Telomerase reverse transcriptase (TERT) activity.

• Contains an Telomerase RNA component (TERC) that
  provides the template for telomere repeat synthesis.

• epeated translocation and elongation steps results in
  chromosome ends with an array of tandem repeats.

with the telomerae the copy gets made adn then added to stop cell death

Regulation of telomerase activity

• telomera length involves accessibility of telomeres to telomerase

• length control factors include

  • proteins POT1, TRF1, TRF2

Telomerase, aging, and cancer

• telomerase Has HOUSEKEEPING function- in most unicellular organisms

• Progressive shortening of telomeres

  • in most human somatic cells not enough telomerase is expressed to maintain length

• High levels of telomerase activity in ovaries, testes, rapidly dividing somatic cells, and cancer cells.

Telomere shortening: a molecular clock for aging?

• Telomerase- target of anti-aging/ or cancer therapy

• in cancer cells

  • telomerase has been reactivated

Telomerase and aging: the Hayflick limit

• The Hayflick limit is the point at which cultured cells stop dividing and enter an irreversible state of cellular aging (senescence).

• Proposed to be a consequence of telomere shortening