Unit 2- DNA Replication

What model organism was used for proks and for euks

• Proks→ e.coli

  • circular small genome- ~ 4000 protein coding genes

• Euks→ Budding yeast

  • Linear, smallish euk genome, ~6000 protein coding genes

  • Strongly homology with humans for essential genes

    • established through complementation studies

Complementation studies for budding yeast show

• When the yeasts essential replication gene mutated this is lethal

• But when human homolog of this gene added, it replaces the yeast gene’s function→ yeast is fine doesn’t die → complementation

• therefore, human gene is ortholog of yeast gene

  • gene that retains same fx across diff organisms

Semi conservative replication model

• Each daughter DNA molecule has 1 original (parental) strand, and 1 newly synthesized strand

• New N atoms into both of its 2 daughter cells

  • Each daughter DNA has 14N and 15N

Conservative replication

• Original DNA stays intact, a completely new double helix is made

• New N atom into one daughter

  • 15N in the daughter that is the og copy, and 14N in the daughter DNA molecule that is new

Dispersive replication model

• Old and new DNA are mixed in fragments within each strand

• both daughters have N

Set up: Meselson Stahl Expt

• Grow E.coli in heavy nitrogen- 15N for 14 gens

  • N bc it’s found in DNA bases

• Everytime a cell divides + DNA replicates it incorporates new N atoms into the DNA of either 1 or both daughter cells

  • depending on which model was correct

After switching E.coli back to 14N…

• DNA seperated using CsCl

• DNA that has 15N will be denser→ move further down the gradient

• DNA with 14N= lighter→ bands higher

• Hybrid DNA (15N/14N) settles in between

After 1 round of replication: Meselson Stahl Expt

• Observed 1 single band at intermediate density (15N/14N)

• Means all DNA molecules are hybrids

• RULES out conservative model because this would produce

  • 1 heavy band (old DNA)

  • 1 light band (new DNA)

After 2+ Rounds Replication: Meselson Stahl Expt

• Observed 2 bands- 1 light (14N/14N) and 1 hybrid (15N/14N)

• Means DNA replication not dispersive bc the expt produced a separate/distinct light band

  • If it was dispersive we would get only 1 hybrid band that lightens

• Matches semi-conservative replication

  • therefore each DNA molecule contains 1 og parental strand and 1 newly synthesized strand→ confirms watson and cricks prediction

E.Coli Bacterial DNA Replication

• Begins at the replication origin (ori)

  • Replication forks

• Proceeds bi-directionally

  • theta-replication→ bc looks like symbol

• Once finished the linked circles are separated by topoisomerase

Is the origin random for bacterial replication (e.coli)?

• No there’s a specific “consensus” sequence

• best characterized bacterial origin= oriC

  • 254 bp, 2 repeating arrays→ 3 ×13-mer and 4×9-mer

  • Sequence AT rich because easier to break 2 H bonds than 3

E.Coli vs Humans

• #bp/cell, rate of rep, # rep origins, mistakes made

Fts	E.coli	Humans
#bp/cell	less	more
Rate of replication	faster	slower
# replication origins	1	many bc there’s billions of base pairs- too big for a single origin
Mistakes made	10^-6	10^-9 (less mistakes per base replication)

Origin consensus sequence euks

• larger and varies a lot b/w euk

  • more varied than proks BUT still AT rich

Yeast origin

• Autonomous replicating sequence (ARS)

• AT rich, 100-150 bp

• 11 bp core consensus sequence

• multicellular euk origins tend to be larger and more variable but also AT rich

Replicons

• segment of DNA that’s replicated from 1 origin of replication

• Each origin is start of a replicon

• replication proceeds bidirectionally from each origin

What happens when replication starts in eukaryotes

• Replication starts→ replication bubbles form→ fusion of bubbles/replicons

• Bidirectional synthesis

  • leading strand-towards rep fork

  • lagging strand- away from rep fork

DNA polymerase synthesizes DNA only..

• 5’→3’

• bc adds nucleotides to 3’OH

Leading strand

• Synthesis proceeds continuously from a single primer

• towards rep fork

Lagging strand/Okazaki Fragment

• Synthesis proceeds in segments (bc DNA poly can only add 5’→3’) requiring multiple priming events

  • =Okazaki fragments

• Goes away from rep fork

Bacterial Initiation

• What’s Dna and SSBs

• Dna =family of enzymes- open DNA

• SSBs= ss DNA binding proteins

  • stabilize the single strands preventing them from zipping back together (reannealing)

Steps of Bacterial Initiation

1. Dna A binds to oriC @ 9-mer repeats

• causes torsional strain which opens 13-mer region

2. SSBs prevent re-annealing

3. Dna C loads Dna B onto the DNA

4. Dna B (a helicase)

• opens helix by breaking H bonds

• Uses ATP hydrolysis

Eukaryotic Initiation steps (to license DNA for Replication)

1. ORC (origin recog. complex)

• Binds to origin, marks where rep begins

2. Helicase loaders→ help MCM (a helicase) bind ORC

3. MCM (minichromosome maintenance complex)

• Several helicases (loaded onto DNA but remain inactive)

All these steps and enzymes= pre-replication complex→ DNA licensed for replication

Helicases

• Unwinds DNA

DNA polymerases

• Catalyze phosphodiester bond

• Synthesize new DNA (5’→3’)

• most accurate enzyme

Topoisomerases

• reduce supercoiling

Primases

• Create RNA primers (3’OH) giving DNA polymerase place to begin replication

• Primase is a RNA polymerase can make RNA primer from scratch unlike DNA poly providing the 3’OH for DNA replication

Ligases

• Seal the backbone of newly synthesized fragments

• glue

Families of DNA polymerase and which used by euks and proks

• By sequence homology- 7 families

  • A, B, C, D, X, Y

• Bacteria use family C for replication

• Euks use family B

• Archae family B and D

Euks, Archae, and Bacteria

• Sliding Clamp

• Clamp Loader

• Sliding clamp- holds DNA polymerase in place on template

  • increases how many nuc polymerase can add w/out falling off

• Clamp loader- loads sliding clamp onto DNA

Organism	Sliding Clamp	Clamp Loader
Euks/Archae	PCNA (measure of how many cells divide)	Rep factor C
Bacteria (E.Coli)	B- Clamp	Y-Complex

Pol a-primase (primase in proks)

• Complex w/ primase makes 20-30 nt RNA-DNA primers

• Provides starting pt by creating RNA primers since DNA poly can only add nucs to 3’ end of nucleotide chain

Pol δ/ε (DNA pol 3 in proks)

• Main replicative polymerases

• each include 3’-5’ proof reading activity

• ε→ leading strand

• δ→ lagging strand

These polymerases work together during replication

All together: (prok) DNA replication (11 steps make a flowchart)

1. Initiator proteins bind to dsDNA

• Slight unwinding

2. DNA helicase- continues unwinding

3. DNA gyrase- relaxes supercoils

4. SBS-stabilizes single strands

5. Primase binds- synthesizes RNA primer

6. DNA poly 3- adds nucs in 5’→3’ direction (ig prok replication)

7. RNA primer made for lagging strand

8. DNA poly 3 extends

9. Discontinuous synthesis of lagging strand- multiple primers, Okazaki fragments formed

10. DNA poly 1 (prok only)- removes RNA primers

11. DNA ligase links Okazaki fragments

Replisome

• entire protein complex that carries out DNA replication (to coordinate bidirectional synthesis)

• This is the “replication machine” that moves along DNA

In eukaryotes

• DNA pol 1 is replaced with..

• Replisomes replaced with…

• DNA pol 1 replaced w/ RNase H (cuts RNA/DNA hybrids) and FEN 1 (RNA exonuclease)

  • (DNA pol 1 removes RNA primer in proks)

• Replisomes replaced w/ replication factors

Additional proteins needed to manage nucleosomes

• chromatin remodeling proteins- loosen nucleosome packing ahead of rep fork

• NAP-1→ (deliver histones safely)

• CAF-1→ (assembles nucleosomes on new DNA)

What is a problem that Eukaryotes have at the end of replication proks don’t

• Euk chromosomes are linear

• Polymerases can only add nucs to the 3’OH of an existing nucleotide

• But when there’s no 3’OH at the end, this end bit can’t be replicated

• Each round of replication makes shorter + shorter DNA molecules

Solution to end of replication problem

• Telomeres

• contain highly repeated DNA sequence (TTA GGG)

• 100-1500 copies

• non coding DNA

Why are telomeres a solution

• They ensure that any loss will not be an immediately important sequence

• Hides the ends of chroms so they aren’t seen as ds break damage

  • T loops

• But when telomeres get too short the Hayflick limit reached + apoptosis triggered

  • Bc the cells recognize the end part as a problem now

Telomerase- maintains telomeres

• Ribonucleoprotein w/ reverse transcriptase activity ( makes DNA using RNA as template)

• Not expressed in all cells (mainly germline, eggs/sperm)

• Provides RNA template to make more repeat sequence

• Recruits capping proteins to protect end of telomeres

  • so it doesn’t look like ds DNA break

Sanger Sequencing (chain termination method) →(innovation came from understanding DNA rep)

• Dideoxynucleotides

• Have a 3’H that prevents chain extension bc no 3’OH

• The color of fluorescent dye on the dideoxynucleotide reps the last base that was added to that fragment

Sanger Sequencing (chain termination method)

• Labelled dNTPs

• Allows determination of terminal base (end-label)

• Radioisotopes→fluorophore (innovation)

Principle of sanger sequencing

• Generates DNA fragments of all lengths which are separated by electrophoresis to det the sequence of a DNA strand