Gene-L7- DNA replication II

major challenges of DNA replication?

1. speed- DNA pol most synthesise DNA rapidly and need to be tightly bound to template DNA

2. end replication problem- chromosome ends cannot be fully replicated due to lagging strand and needs telomeres and telomerase

what are the general features and requirements of DNA polymerase? what does it need? what does it associate with? what are the substrates?

• substrates: dATP, dGTP, dTTP and dCTP

• DNA polymerase is template directed and needs a primer with a 3’OH group and is later extended with DNA

• high efficiency depends on staying attached to DNA

• associated with sliding clamp proteins:

• ecoli B2 subunit

• humans PCNA

mechanism of DNA polymerase catalysis during nucleotide addition

• 3’ OH of growing DNA forms a nucleophilic attack and attacks phosphate group of incoming dNTP

• forms covalent phosphodiester bond and releases energy phosphate groups and AT CG occurs

what are the key properties of e.coli DNA polymerases?

• 3 polymerase I, II, III

• all have proofreading 3’ to 5’ exonuclease- checks last nucleotide and hydrolyses it if wrong

• some have 5’ to 3’ exonuclease

• for error correction, primer removal, gap filling and DNA repair

key features of DNA pol III in e.coli? what are the key structural components?

• main replicative enzyme

• very efficient- 50 nucleotides per second

• works on both strands

• large multi polypeptide complex

• core polymerase: 3 subunits

1. alpha- nucleotide addtion

2. epsilon- 3’ to 5; exonuclease and proofreading

3. teetha- stabilises epsilon

4. beta/gamma- convert enzyme from disturbutie enzyme(easily fall off) to processive enzyme

how does pol III function at replication fork? in e.coli

1. clamp loader gamma complex

• loads sliding clamp onto DNA ATP dependent and also unloads

• t dimerises 2 core polymerases- coordinates leading and lagging

ATP opens sliding clamp and placed on DNA- pre initiation complex

pol III binds and initiation complex which starts synthesis

• Pol I- removes RNA primers 5’ to 3’ exonuclease

• Pol II- DNA repair error prone

• Pol III- main replication enzyme

what are the main eukaryotic DNA polymerases and their roles? 3

Pol alpha- initiation polymerase

• very large enzyme and works with primase to make RNA primer

• catalytic activity and no sliding clamp association

• not highly processive

pol delta- lagging strand synthesis.

• associated with PCNA and has proofreadingg activity

• extends primers by pol alpha and primase

pol epsilon-leading strand synthesis

• DNA pol of leading strands and extends the primer

• can proofread and highly processive

how does polymerase switching and clamp loading work in eukaryotes?

• primase and Pol alpha synthesis RNA primer and short DNA stretch

• replication factor C- loads sliding cam using ATP

• switch occurs- poly delta or epsilon bind via PIP motifs and extend DNA depending if its lagging or leading

• sliding clamps: PCNA 3 subunits and E.coli Beta clamp dimer- increase processivity

how is DNA replication terminated in eukaryotes vs prokaryotes?

• it happens when 2 replication forks meet

• RNA primers are removed and replaced by DNA. RNase H removes RNA primer and FEN1 removes remaining flap. E.coli has Pol I with 5’ to 3’ exonuclease activity

• pol delta lagging and pol epsilon leading fills gaps with DNA

• remaining nicks are sealed by DNA ligase I

• replication machinery disassembles

• MCM helices is ubiquitinated and removed

what ar key proteins and steps in eukaryotic DNA replication? 6 steps

1. imitation: ORC proteins recognise origins. CDC6 and Cdt1 load MCM helices and licensing factors

2. unwinding- MCM helices opens DNA. SSB proteins stabilise ssDNA and topiosiomerase relieves supercoiling

3. priming- pol alpha and primas synthesise RNA primer and short DNA

4. elongation- Pol epsilon leading and pol delta lagging. use PCNA for high processivity

5. primer removal- RNase H and FEN1 remove RNA primer- leave a nick

6. DNA ligase I seals nicks

what happens at the end of DNA replication in eukaryotes? how is this prevented?

• removal of last RNA primer on lagging strand leaves a DNA gap and chromosome is shortened

• telomerase at chromosome ends TTAGGG G rich and forms a terminal overhang structure to prevent loss. without telomerase progressive chromosomes shortening

what are telomerase and what affects the length?

• functions to protect chromsome ends and prevent DNA damage response activation and stop end to end chromosome fusion

• telomere shortening is linked to cell ageing- somatic cells have no tolerates so they gradually shortened

• factors that affect it are cell division and shortens with age, oxidative stress accelerates shortening