Genetics Chapter 9

Replication of DNA

Replication must be extremely accurate

Must take place at high speed

Semi-conservative

DNA replication is Semi-conservative

The original parent strand splits and synthesizes a new strand

• Parent strand (2 parent strands)

• 2 daughter strands (each contain 1 parent strand one new strand)

Meselson and Stahl’s experiement

Discovered DNA is semi conservative

Experiment consisted of using N14 and N15 (Heavy Rare)

E. coli grew in N15 first than transferred to N14 after

N15 heavier sinks lower after centrifuged and N14 was lighter so was higher

Replicon

A unit of DNA that is replicated from a single origin of replication.

• One origin

• Plus all the DNA that gets copied from that origin

• = one replicon

Prokaryotic Cells have 1 Origin of replication

Eukaryotic Cells have thousands of origins

Different types of Replication

• Theta (θ) replication – circular chromosomes (prokaryotes)

• Rolling-circle replication – plasmids (some viruses)

• Linear (Eukaryotic) replication

What do all DNA replication modes need?

• Template strand

• Raw material: Nucleotides

• Enzymes and other proteins

• Source of energy

Bacterial replication

Circular DNA template

DNA opens up at the origin of replication

Replication bubble is formed and replication begins to occur bidirectionally

When replication reaches termination region it ends

Bacteria chromosome has ONE replicon

Results in 2 circular DNA strands

Which way does DNA replicate?

All Replication (Prokaryotes & Eukaryotes) involves adding to the 3’ end of the growing chain

It grows 5’ → 3’ direction

DNA polymerase can only add nucleotides to a free 3’-OH group

Leading strand

One long continuous replication strand

The 3’ end is facing the inside of the replication fork

As the fork unwinds more nucleotides are added to the 3’ end

Lagging strand

Discontinuous replication several small segments (Okazaki fragments)

The 3’ end is facing away from the fork

As the replication fork unwinds DNA synthesis has to keep beginning again

Okazaki Fragments

Short DNA fragments that produced by the discontinuous DNA synthesis on the lagging

Helicase

Is an enzyme that unwinds DNA at the replication fork

Topoisomerase

relieves the supercoiling ahead of the replication fork

Deoxynucleotide triphosphates (dNTP)

What new nucleotides first come in as

A base

A sugar

and Three phosphates

3’- OH When nucleotide is added to DNA

The 3’ - OH attacks the 5’ phosphate group of the incoming dNTP and two phosphates are cleaved off

This forms a covalent bond

(Only occurs if the correct base pair is added) (A-T, G-C)

Pyrophosphate

The two phosphates that break off are pyrophosphate and this releases energy

This energy is what pushes DNA synthesis forward and the energy for the covalent bond to form

Stages needed for DNA replication

Stage 1: Initiation

Stage 2: Unwinding

Stage 3: Elongation

Stage 4: Termination

Stage 1: Initiation

The goal is to open DNA and assemble replication machinery

Replication starts at Origin of replication

DnaA monomers bind to the origin of replication and initiates unwinding

Stage 2: Unwinding

DNA helicase binds to lagging-strand and unwinds DNA moving in the 5’-3’ direction breaking hydrogen bonds moving the replication fork

Single Stranded binding proteins keep exposed DNA stable

DNA gyrase relieves strain ahead of replication fork

Initiator protein

Binds to origin and separates strands of DNA to initiate replication

DNA helicase

binds to lagging-strand and unwinds DNA moving in the 5’-3’ direction breaking hydrogen bonds moving the replication fork

Single-stranded binding proteins

Stabilize the exposed single-stranded DNA and keeps it stable during DNA replication

DNA gyrase

Relieves strain ahead of the replication fork

Moves ahead of the replication fork, making and resealing breaks in the double-helical DNA to release the torque that builds up because of unwinding at the replication fork

Stage 3: Elongation

Synthesizes new DNA strands

DNA Polymerase III adds a nucleotide in the 5’ → 3’ direction

A free 3’ - OH group is needed in order for a new nucleotide to be added

DNA Polymerase III

Is what synthesis the new DNA strand by adding new nucleotides on to the 3’ OH group in the 5’-3’ direction

DNA Polymerase III cannot initiate DNA synthesis on a bare template

DNA Polymerase III initiates on an RNA primer

Also proofreads for any mistakes in the new synthesized DNA strand

DNA polymerase I

Removes the RNA nucleotides of a primer and replaces it with DNA nucleotides

Removes rNTPS replaces with dNTPS

Primase

Primase is a RNA polymerase and does NOT require a 3’ -OH group

Lays down RNA primer that provides a 3’ -OH group to which a new nucleotide can be added by DNA polymerase III

RNA Primer

Small segment of RNA nucleotides that provides a 3’-OH group so that DNA Polymerase III can add a new nucleotide

DNA Ligase

Joins Okazaki fragments by repairing nicks in DNA backbone by catalyzing phosphodiester bonds

These nicks are created after DNA polymerase I replaces RNA with DNA there still remains a nick

Stage 4: Termination

This happens when two replication forks meet or by a specific termination sequence

How eukaryotic DNA replication Differs from prokaryotic DNA replication

More complex Tertiary structure (Packing of DNA into chromosomes)

Chromatin must relax prior to replication

Eukaryotes have many more DNA polymerase than bacteria

The linear structure of eukaryotic chromosomes creates a problem with replicating ends of chromosomes

Origins of replication (Eukaryotic DNA)

There are multiple origins of replication

Initiation requires two steps

• Licensing of the origins by licensing factors

• Initiation of replication at each licensed origin

DNA Polymerase Alpha (Eukaryotic)

Has the same job as primase

Creates an RNA primer so that elongation can occur

DNA Polymerase Delta

Polymerase activity in the 5’ to 3’ direction on the lagging strand synthesizing new DNA strand

DNA Polymerase Epsilon

Polymerase activity in the 5’ to 3’ direction on the leading strand synthesizes new DNA strand

The End of Replication Problem

Every cell division a little DNA is lost, chromosomes become shorter each generation and eventually destabilize and are degraded

Telomerase

Is a Ribonucleoprotein enzyme (protein + RNA) that extends telomeres

The ends of chromosomes are replicated and extended

Telomerase attaches to overhanging 3’ end than its RNA pairs with the telomere DNA and adds new DNA

(Rebuilds what replication removes and preserves chromosomes length)

Protective function of Telomeres

the repetitive sequence at the end of chromosomes relives impacts of degradation by forming protective T-loops the protect against degradation