DNA Replication

DNA STRUCTURE

• Double stranded

• 4 bases: ATCG

• Double stranded

• Anti-parallel

• 3 parts to the nucleotide:

  • Deoxyribose

  • Phosphate group

  • Nitrogenous base

ANTIPARALLEL
5’ end is where the phosphate group is

3’ end is where the sugar ends

From top to bottom, one strand is 5’-3’ and the other strand is 3’-5’ (because it is upside down)

UNIFORM WIDTH

A pyrimidine (1 ring)  is paired with a purine (2 rings) in order to maintain a uniform width.

• C-G has 3 bonds

• A-T has 2 bonds

DNA REPLICATION

Semi-conservative

Replication will have one original strand and one new strand.

The original strand is conserved, but it is only one of them, so it’s semi-conserved

ENZYMES USED IN REPLICATION

• Helicase - enzymes that untwist the double helix at the replication fork & separates the two parental strands

• Topoisomerase - enzyme that relieves the strain caused by the untwisting of the strands (don’t unwind too far)

• Primase - synthesizes RNA primers, using the parental strand as a template (both strands)

• Single-strand binding proteins- bind to unpaired DNA strands and help stabilize them

ENZYMES involved in REPLICATION

• DNA polymerase I and III-  adds nucleotides to the growing DNA strand; 

  • DNA polymerase ONLY moves in the 3’ to 5’ direction

  • Nucleotides are only added to the strand on the 3’ end 

  • Causes a leading and a lagging strand to form because of the antiparallel structure of DNA

• Ligase - joins the sugar-phosphate backbones of the Okazaki Fragments

LEADING STRAND

1. Primase makes an RNA primer at the start of the DNA strand (the 3’ end).  DNA Polymerase cannot build a strand without a nucleotide to build from

2. DNA polymerase III then moves from the 3’ to 5’ direction on the parent strand building from the 3’ end of the primer.  Strand is built 5’ to 3’.

3. DNA Polymerase can simply follow helicase and create the strand directly

LAGGING STRAND

DNA polymerase must create the new strand in the 5’ to 3’ direction because it can only add nucleotides to the 3’ end.

Because DNA is antiparallel, that creates a traffic jam at the replication fork.

• This strand is formed discontinuously in a series of segments called Okazaki Fragments

• Each fragment requires its own primer

• DNA polymerase III  forms the Okazaki fragments.

• DNA polymerase I replaces the RNA primer with DNA nucleotides

• Ligase then combines the fragments together

Telomeres  

At the end of the parental strands, some of the DNA cannot be replicated.  

Telomeres – in defense of this DNA shortening, there are repetitive segments at the end of DNA strands that protect the actual coding sequences 

Remember that there is a primer at the 5’ end of every DNA strand.  This RNA primer provided a starter for DNA Polymerase III.

When the primers are removed, there are no nucleotides for the DNA polymerase to use as a base for replication attachment.  So those overhangs will not be replicated and slowly subtract from the telomeres.  

Helicase begins by opening up the DNA strands.  This is now the replication fork at the point of origin.

As helicase opens the strand, primase adds a RNA primer in the 5’ to 3’ direction (moving 3’ to 5’ on the original strand).  Remember, DNA polymerase III cannot begin a strand on its own – it must attach nucleotides to an already existing strand.

Once Primase has laid down the primer, DNA polymerase III begins attaching DNA nucleotides off of it.  

As helicase moves along the strand, it opens up more nucleotides to be replicated.  

For the leading strand, DNA Polymerase III can continuously add nucleotides as it follows helicase.

For the lagging strand, there are now nucleotides available in front of the primer that were not available before (the starred area) 
Primase is now needed again to create a new primer for the newly exposed DNA nucleotides on the lagging strand.

Once the primer has been placed, DNA Polymerase III can replicate the DNA until it reaches the primer from earlier.  

This process continues…helicase opens up more DNA nucleotides, a primer is produced by primase, and DNA Polymerase III adds nucleotides until it runs into the RNA primer from before.

As the process continues, the DNA fragments that are found between the RNA primers are called Okazaki Fragments.  These are short strands of DNA found on the lagging strand during replication.  At this point, the RNA primers need to be replaced with DNA and the strands need to be sealed/bonded together. 

The RNA Primers are then replaced be DNA Polymerase I using the previous Okazaki fragment as its starting point.  Now that the strand is all DNA nucleotides, ligase comes and seals one Okazaki fragment to the next.  This causes the little nicks between fragments to disappear and the strand becomes continuous.

Proofreading & Repairing DNA

As DNA is being replicated, there can be errors.  

In order to fix the errors, there are several options.

1. Proofreading

2. Mismatch Repair

   1. Base excision repair

   2. Nucleotide excision repair

3. Double strand break repair

The most efficient way to fix DNA errors during replication is for DNA Polymerase to proofread the nucleotides as it moves along the strand.  DNA Polymerase III has the ability to remove and replace the incorrect base pair during replication.

Mismatch Repair

Mismatch repairs happen right after replication.

They can replace incorrect nucleotides missed during proofreading & they can remove slight insertions/deletions when DNA Polymerase “slips” during replication.

This is done through two mechanisms:

1. Base excision repair

2. Nucleotide excision repair

Base Excision Repair

• When there is an incorrect base, a glycosylase removes the incorrect base, but leaves the sugar-phosphate backbone.

  • There are specific glycosylases for each nucleotide

• The sugar-phosphate backbone is then removed by other enzymes.  

• DNA Polymerase then fills the space with the correct nucleotide.

Another common result of DNA damage is with cytosine.

If it loses an amino group, it becomes uracil.  This needs to then be replaced.

This is an example of base excision repair.

Nucleotide Excision Repair

The damaged nucleotide is removed along with the surrounding nucleotides.

DNA Polymerase replaces the nucleotides and ligase connects the segments.

Some common DNA errors

UV damage can cause two TT nucleotides to covalently bond together instead of forming a hydrogen bond with the As on the other strand.

This is an example of nucleotide excision repair

Double Strand Break Repair

These are dangerous because if the break is not repaired, those genes will be lost.

This type of damage usually occurs after heavy radiation such as gamma or x-rays.