BIO2089 Central Dogma & DNA Replication

Central Dogma

• How info is stored in DNA

• faithfailly maintained

• expressed through transcription and translation

• how those processes are replicated

DNA is used over RNA because its less

chemically reactive, it lacks an 2’OH group so more stable

DNA is used over RNA because its less

prone to mutate, C can be spontaneously deaminiated to U which wouldn’t be detected in RNA

Genetic material must be

• Stable over time

• able to be faithfully replicated

Information flow

1. RNA world, RNA = info carrier

2. RNA produced proteins (translation)

3. DNA replaced RNA as genetic material (replication & transcription)

dNTPs (deoxyribose nucleotide triphosphates)

substrate for producing DNA

Pyrimidines

C, U, T

Purines

A, G

DNA contains

• Nitrogenous base

• 5 carbon sugar

• Phosphate groups

What provides energy for DNA synthesis

breakage of phosphate groups & release of pyrophosphate

DNA structure

double stranded (antiparrallel) w/ H bonds

H bonds between G & C

3

H bonds between A & T

2, less energy required to break them

Proteins bind within the

major grooves of DNA in a sequence specific fashion

DNA is the

polymer

Nucleotides are the

monomer/building blocks = sugar, base & phophate

Dispersive theory of replication

DNA wouldn’t be synthesised in 1 long chain, short regions would be replicated & stiched together (new & old bits)

Conservative theory of replication

Parental DNA will stay together and newly sythesised DNA would stay together

Meselson & Stahl

1. Ecoli grown on 15N (heavy)

2. Cells switched to 14N (light)

3. Sample after 20 min (1st replication)

4. Sample after 40 min (2nd replication)

Replication fork

location where the 2 strands of DNA are unwound during replication & new DNA is being synthesised on the basis of base pairing

DNA repliaction is

bi-directional from the origin of repliaction

How many active replication forks are there

2 going off in opposite directions

Bacteria have __ origin of replication with __ replication fork

1 with 2 (theta structure)

Eukaryotes have __ origin of replication with __ replication fork

multiple with each replication bubble having 2 forks

What is needed for replication

• DNApol 1 + Mg cofactor

• dNTPs

• ssDNA template

• Primer 3’OH (DNApol needs to extend from a 3’OH)

Direction of replication

5’ to 3’

DNApol1 activites

• synthesise DNA

• 3’-5’ exonnuclease - proofreading

• 5’-3’ exonnuclease - RNA primer removal

DNApol1 is

not major pol in replication as mutants in polA are viable

DNApol3 is

major pol as mutants in dnaE gene are inviable

Leading strand syntheis

continous, 3’-5’

Lagging strand synthesis

discontinous, okazaki fragments, 3’-5’

Okazaki fragments are longer in

bacteria than in eukaryotes

DNA helicase is recruited to

orgin of replication to unwind dsDNA

DNA helicase acts

5’-3’ on lagging strand to dissociate leading strand

DNA helicase is

ATP dependent

How does DNA helicase move along ssDNA

hydrolyses ATP when bound to ssDNA

In bacteria DNA helicase

assembles as a homohexamer ring

ss-binding proteins

stabalises DNA by binding cooperatively to ssDNA to prevent secondary structures (hairpins & stem loops) which keeps DNA accessible

Topoisomerase function

relieves overwinding of DNA happening when the region infront of unwinded DNA undergoes postive supercoiling

Topoisomerase 1

introduces ss breaks allowing DNA to rotate around the 1 phosphate keeping it togteher then rejoins DNA

Topoisomerase 2

introduces ds breaks to pass through a strand of DNA through the break, rejoins DNA —> decatenates chromosomes

Primase

synthesises primer = RNApol

Primer length in bacteria

10 nts

Primer length in eukaryotes

10 nts of RNA then 10-20 nts of DNA

Primer synthesis in eukaryotes

complex of primase enzyme to synthesise RNA nts and DNA pol a to add DNA nts

Primers initate synthesis by

joining 2 NTPs

Primer activity is

increased in presence of helicase

RNase H

removes primer & trims okazaki fragments by recognising RNA associtaed with DNA

what can RNase H not do

break the bond between the RNA molecule & DNA nt, cant break the last residue of the primer - this is done by DNA pol 1 which then completes synthesis across gap

DNA ligase

Joins the DNA backbone using energy from ATP to drive ligation, joins okazaki fragments

DNA ligase mechanism

1. Hydrolyses ATP to activate the 5’ phosphate of 1 fragment

2. Pyrophoshate released & AMP is covalently attched to phosphate group

3. 3’OH residue attacks 5’ phosphate, breaks the phosphate group & forms a new bond with the 3’OH of the other fragment

4. DNA backbone is reformed & AMP released

Sliding DNA clamp forms

a ring around DNA to prevent the release of DNA pol = increasing processivity of DNA polymerase = ensures DNA pol remains held in correct site associated with DNA

Sliding DNA clamp is not immediately released after replication because

it recruits other enzymes to finish replication

Clamp loading

1. Clamp loader + ATP = binds to sliding clamp & breaks ring of the sliding clamp

2. Sliding clamp recruited onto DNA & clamp loader associates with DNA

3. Recognition of primer template junction activates ATP hydrolysis (clamp loader ATPase) causing release of clamp loader & closing of sliding DNA clamp ring around DNA

4. DNA pol binds to sliding clamp

Replisome

number of enzymes that act in complex to replicate/synthesise DNA

Bacterial DNA pols for DNA repair

pol 1, pol 2, pol, 4

Bacterial DNA pols for RNA primer removal

pol 1

Bacterial DNA pols for chromosome replication

pol 3

Bacterial DNA pols for translesion synthesis

pol 5

Eukaryotic DNA pols for DNA replication

alpha, y (mitochondria) , delta (lagging strand), E (leading strand)

DNA synthesis step 1

primase synthesises short RNA oligonucleotides (primers) copied from DNA

DNA synthesis step 2

DNA pol 3 elongates RNA primers with new DNA

DNA synthesis step 3

DNA pol 1 removes RNA at 5’ ed of neighbouring fragment & fills gap

DNA synthesis step 4 (final)

DNA ligase connects adjacent fragments

Replicon model

1st model for how initation of replication could be achieved

Replicon

the region of DNA that’s associated with a particular origin of repliaction

Bacteria replicon

entire chromosome (plasmids = seperate replicons)

Eukaryotes replicon

multiple replicons build up the chromosome

origin of replication

site where unwinding of DNA & initation of replication occur

Replicator

cis acting site sufficient for replication, origin of replication is part of it

Initiator

Protein(s) that recognise replicator & initiates replication & recruits machinery for DNA synthesis

Bacteria initator

DnaA

Bacterial replicator has 5 copies of 9-mer sequnece

DnaA binding sites/boxes

Bacterial replicator has 3 copies of 13-mer sequnece

AT rich region (less energy to unwind DNA, where unwinding starts)

DnaA in the presence of ATP

binds to 9-mer reapeats = DnaA boxes

DnaA bound to DNA causes

localised unwinding of AT rich region & helicase loader recruited to load helicase to origin & helicase loader replaced by DNA primase

Regulation of bacterial DNA replication step 1

dam gene encdes dam methylase that methylates A in the GATC sequence of which there are 11 in oric (orignin of replication)

Regulation of bacterial DNA replication step 2

After replication DNA is hemimethylated (parental strand methylated, new strand not)

Regulation of bacterial DNA replication step 3

SeqA binds hemimethylated DNA & blocks recuitment of DnaA (initator)

Regulation of bacterial DNA replication step 4

Seq A not permentalty bound, when not bound A is methylated & DnaA can bind to initate replication

Eukaryotes DNA replication limited to the

S phase of the cell cycle

During S phase an orgin of replication is only allowed to fire

once

Eukaryotic replicator in yeast

Yeast ARS (autonomously replicating sequneces), well definied

Eukaryotic replictor in higher eukaryotes

not well defined, zones of initation, cover larger regions

Eukaryotic initiator

origin replication complex (ORC)

origin replication complex (ORC)

6 protein complex

What happens in G1 phase

1. ORC binds to origin

2. ORC recruits helicase loading proteins (cdc6 & cdt1) which recruit helicase (mcm2-7) & hold it inactive = prereplicative complex

What happens in S phase

1. cdc6 is phosporylated via cdk mediated phosphorylation

2. helicase loading proteins dissociate

3. Helicase becomes active & unwinds

4. phosphorylated cdc6 degreaded

5. Recruitment of replication machinerary

6. phosphorylation of ORC = no more helicase binding proteins can bind

Decatenation of chromosomes

after replication chromosomes linked together in a chain so need unlinked to be able to go into different cells

The ends of lagging strands are

single stranded & recognised by telomerase

Telomerase

composed of protein similar to reverse transcriptatse & RNA which is the template for DNA

Telomerase mechanism

1. binds to single stranded region of chromosome (RNA element & end of telomere DNA)

2. extends 3’ end (reverse transcriptase)

3. goes back & adds the same repeat multiple times to protect the chromosome

4. completion of lagging strand synthsis by DNA pol

chromosome ends form a t loop beacause

action of telomerase still leaves ssDNA region

formation of a t loop

chromosome folds back on itsself & ss region undergoes strand invasion & base pairs with main strand of chromosome & 3’OH hidden from degredation

telomere binding proteins

recognise telomers & bind ti flag them as telomers

DNA replication accurate because of

• DNA pol base pairing

• DNA pol proofreading

• strand directed mismatch repair

DNA pol is dependent on

base pairing specificity at the active site DNA

DNA pol mispairing is

transient, causes distortion of helix & is resolved

tautomeric forms of bases

rare spontaneous chemical conversions can base pair without causing distrortion to helix but are lost because they are unstable

DNA pol checks correct base is added

1. before breaking phosphate group

2. before releaseing pyrophosphate