DNA Replication

Processing of Genetic Material

What is DNA Replication

• The process by which the DNA within a cell makes exact copies of itself

• Balance of speed and accuracy• Used for growth, repair and embryonic

development• Done during Interphase of mitosis

When…. S phase

Possible ModelsConservative – original intact and a completely new daughter DNA

Dispersive Model-a mixture of old and newly synthesized DNA parts

Semiconservative -   Both daughter strands are composed of an old strand and one new

Isotopes were used to show that DNA replication was semiconservative 

Messelson and Stahl

DNA Replcation

• DNA replication is semiconservative

• The parent double helix produces 2 daughter double helices.

• Each daughter molecule will have a parental strand and a daughter strand (an old strand and a new strand)

A nucleoside consists of a nitrogenous base covalently attached to a sugar (ribose or deoxyribose) but without the phosphate group. A nucleotide consists of a nitrogenous base, a sugar (ribose or deoxyribose) and one to three phosphate groups. Nucleoside = Sugar + Base Nucleotide = Sugar + Base + Phosphate

*A nucleoside monophosphate = nucleotide

Nucleoside vs. Nucleotide

The Process

• Overview– Double helix is unwound to expose bases for new

base pairing– Two new strands are assembled using the parental

DNA as a template– New strands reform into helices

The Process

1. The following proteins bind to the replication origin (1 origin for a plasmid, multiple for linear DNA in eukaryotes)

2. The point at which the DNA pulls apart is known as the replication fork (bubble)

3. DNA helicase pulls strand segments apart and by breaking the hydrogen bonds (just ahead of the replication fork)

4. Single stranded binding proteins (SSBPs) keep them from re-annealing.

5. DNA Gyrase – relieves tension

Replication Forks

• DNA synthesis occurs at numerous different locations on the same DNA molecule (hundreds in a human chromosome).

• The replication rate of eukaryotic DNA is 500 to 5000 base pairs per minute

• A human cell typically requires a few hours to duplicate the 6 billion base pairs

Replication fork

• DNA will replicate small segments of the larger strand at a time.

• So only small segments will be unwound and separated by helicase at any give time.

• These segments are called replication bubbles.

• The junction where the 2 strands are still attached is called the replication fork

Helicase

SSB protein

DNA polymerase III

RNA primase

Building via complimentary base pairing

• The new strand of DNA will build using the parent strand as a template

• Done by complimentary base pairing; Remember A-T and G-C

• The free floating nucleotide triphosphate used for synthesis are ATP, GTP, CTP and TTP.

• Two of the phosphates will be removed when the nucleotide is attached to the growing chain of new DNA. This provide energy for sugar phosphate bonds.

Adding Nucleotides

• RNA Primase lays RNA primer that tells the cell where to start adding nucleotides

• The enzyme primase creates an RNA primer – which is 10 - 60 RNA nucleotides.

• The RNA primer temporarily attaches to the 3’ end of a DNA strand.

• The purpose of the primer is to create a starting point for the DNA nucleotides to attach

Adding Nucleotides

• DNA polymerase III attaches new nucleotides to the 3’ hydroxyl end of the parent chains after the primer (5’ of daughter)

• Replication of new strand can only synthesize in the 5’ to 3’ direction

• A primer must be available to serve as a starting point for the attachment of new nucleotides

5’3’

• DNA is always synthesized in the 5’ -3’ direction .

• This means: a nucleotide that is being added will bond its phosphate group (at the 5’ end) to a nucleoside that is already apart of the strand.

• The next nucleotide will bond to the 3’ end of the previous nucleotide with its 5’ end.

Where will the RNA primer attach?

Leading/Lagging• The open 3’ end of the parent strand is known

as the LEADING STRAND can be copied continuously

• The 5’ end is known as the LAGGING STRAND and is copied dis-continuously

LAGGING STRAND• The other strand is called the lagging strand. • It is synthesized discontinuously in the

direction away from the replication fork and in the opposite direction of helicase.

• As a result, short fragments (1000-2000 nucleotides in length) are produced called Okazaki fragments

• (at the beginning of each Okazaki fragment there will be a RNA primer)

Which is the leading strand/lagging?

Okazaki Fragments• Sections copied discontinuously are called

Okazaki Fragments• Primer must be laid at intervals as the fork

reveals more of the parent strand to create the correct direction of replication 5’ to 3’

• DNA polymerase I removes the primers in the lagging strand and inserts proper nucleotides

• DNA ligase joins fragments by linking the sugar phosphate backbone (makes phosphodiester bond)

As it opens new primer is laid and synthesis occurs 5’ to 3’ until it meets the

last primer

Proofreading

• DNA polymerase proofreads the DNA and inserts correct base pairs if any mistakes have occurred

• As the 2 new double strands of DNA are made, they will automatically twist into a helix.

Proofreading

• Errors occur 1 in every 10 000 to 100 000 bases

• DNA polymerases check for correct hydrogen bonding

• If there is a mistake polymerase excises it and inserts correct one

• Brings errors down to 1 in a billion base pairs

Issues

• Each time the DNA gets shorter -100bp each time

Senescence (Aging)

Telomeres• Non-coding, highly repetitive sequences on

the end of DNA• Rich in Guanine nucleotides• Repeated several thousand times• Protect against the loss of genetic material• Telomerase replenishes telomeresEnd replication problem• http://www.youtube.com/watch?v=MjfIWzufq_IHow to reverse aging• http://www.youtube.com/watch?v=DV3XjqW_xgU

OVERVIEWEnzyme/Protein Function

Helicase •Cleaves and unwinds short section of DNA ahead of the replication fork

Single stranded binding proteins SSBs •Keep DNA from re-annealing

DNA polymerase III •Attaches nucleotide to 3’ end of parent strand•Synthesis 5’ to 3’•Proofreads base pairing

DNA polymerase I •Removes RNA primers, proofread

DNA ligase •Catalyzes formation of phosphate bridges to join Okazaki fragments

Primase •Synthesizes RNA primer to begin elongation

Telomerase •Replenishes lost nucleotides in telomeres

Overview

Videos/animations

• http://www.mcb.harvard.edu/Losick/images/TromboneFinald.swf

• http://www.johnkyrk.com/DNAreplic.swf• http://www.andrew.cmu.edu/user/berget/Ed

ucation/TechTeach/replication/DNARepOverview.mov

Online tutorial

• http://www.bioteach.ubc.ca/TeachingResources/MolecularBiology/DNAReplication.swf

• http://www3.interscience.wiley.com:8100/legacy/college/boyer/0471661791/animations/replication/replication.swf

• http://www.wiley.com/college/pratt/0471393878/student/animations/dna_replication/index.html

Online quiz/games

• http://www.funtrivia.com/playquiz/quiz18993515bfd10.html

• http://nobelprize.org/educational_games/medicine/dna_double_helix/

• http://www.syvum.com/cgi/online/tgamem.cgi/squizzes/biology/dna/BioThree.tdf?0