DNA replication and Cell cycle

Who first discovered the cell

Robert Hooke 1665

Schwann 1839: “ the cell is the unit of structure, physiology and organisation in living things”

Who discovered that all cells come from existing cells?

1855- Rudolph Virchow

What did Fleming identify

1882- First descriptions of mitosis, identifying stages and interphase vs mitosis

What did the cell cycle look like until 1950’s

1950s radioactivity experiments

1950’s - used radioactive phosphorus

• found that while the quantity of RNA and proteins increased steadily during the cell cycle, DNA synthesis only happened at one point during interphase

• Divided interphase into G1(46 single chromosomes), S (each chromosome is 2 chromatids) and G2

What 3 models were proposed for replication

1. Conservative

2. Semi-conservative

3. Dispersive

Meselson and Stahl procedure

• used E.coli bacteria to model, grew it in a medium containing heavy isotope N15

• E.coli took this up and used it to synthesise DNA

• Repeated until all the E.coli contains N15

THEN

• bacteria switched to a medium containing N14

• DNA made after the switch would’ve only had N14 available for DNA synthesis

• Took samples after each new generation

Meselson and Stahl results

• used a density gradient centrifuge

• Separates DNA into bands by spinning at high speeds

• Saw that DNA replicated semi-conservatively

• Each DNA strand serves as a template for a new one

1 gen = 100%

2nd gen = 50:50

3rd = 75 N14 & 25 N15

DNA synthesis requirements

• DNA polymerase + Mg2+

• DNTPs (building blocks and energy)

• Template DNA

• Primer 3’-OH

DNA synthesis = 5’→3’

What are dNTPs

Molecular building blocks (A, T, C, G) used in DNA synthesis

• base

• Deoxyribose sugar

• 3 phosphates

Breakage of dNTP phosphodiester bond provides energy for polymerisation

Initiation: prokaryotes

• bacteria have circular DNA & plasmids

• Each chromosome or plasmid has an ‘origin of replication’- a special sequence where it begins

• Bidirectional replication

• Theta structure

• Makes 2 circular DNA molecules

Initiation: eukaryotes

• chromosomes are much longer

• Each will have many origin

• Bidirectional replication creates ‘bubbles’ of replications

Elongation: clamp loader

• challenge = loading and holding onto DNA

• A ‘clamp loader’ loads the sliding clamp onto DNA

Elongation: twisting

• helicase unwinds double stranded DNA using ATP

• This causes supercooling (twists) which need to be unwound

• Topoisomerases unwind the twists

• They break and reform the phosphodiester bonds to untwist the DNA

Elongation: single stranded binding protein

• single stranded DNA forms H bonds with itself

• Single-stranded binding protein prevents this

• Monomers bind but bases are left exposed

Leading vs lagging synthesis

• replication can only happen from 5’ to 3’

• Laggin strand synthesis = discontinuous

• Primase adds lots of RNA primers(oligonucleotides) as it unwinds, and DNA polymerase synthesises sections= Okazaki fragments

• DNA ligand will then join the fragments together

= semi-discontinuous

Elongation: primase

Challenge on lagging stand is that DNA polymerase requires an OH overhang.

Primase synthesises short RNA primers about 10 nucleotides long with the OH overhangs

Elongation: DNA hloenzyme

•DNA polymerase holoenzyme is an asymmetric dimer that catalyses both leading and lagging strand synthesis.

•It synthesises 1000 bp/second

•100 turns of the helix each second.

Double stranded breaks

Chromosomes damages with double-stranded breaks are repaired by:

1. End joining→ joins two ends making a small deletion

2. Homologous recombination→ copies a similar sequence to repair DNA

A double stranded break at the end of a chromosome would result in them being joined together = cell would likely die

Telomere structure

• 3’ overhang of 100s of the same short DNA sequence = 5’TTAGGG-3’ in humans

• T-loop (overhang tucks in/hides) protects the ends from the DNA repair pathways for double stranded breaks

• Shelterin- a protective protein protects the ingle stranded DNA against other repair mechanisms

Telomere replication

1. Binding of telomerase to the overhang 3’

2. Synthesis of new telomere DNA using telomerase RNA as template

3. Telomerase movement to 3’ end of Newley synthesised telomere DNA

4. Synthesis of new telomere DNA

5. Telomerase laves and chromosome ends

6. DNA polymerase completes the lagging strand

Maintaining DNA integrity

1. DNA polymerase can also ‘proofread’

• if an incorrect base is added, DNA only erase can remove it

• Polymerisation continues

2. Mismatch repair

• if an incorrect base is added, mismatch repair detects a bump in the helix

• Proteins repair mismatches in DNA involving MutS and MutL proteins