DNA STRUCTURE & REPLICATION

Last class...• We walked through the historical time line of

the discovery of DNA• We ended with James Watson, Francis Crick,

and Rosalind Franklin

• Franklin was the first to capture an image of DNA– But it was Watson and Crick who finally

discovered the true structure

Watson and Crick’s structure• Double helix that is comparable

to a ladder.

– The uprights are made of:• DEOXYRIBOSE sugars connected by

PHOSPHATE groups• Held together by phosphodiester

linkages

– The ladder rungs are made of:• NITROGENOUS bases

A Closer Look...

Deoxyribose Sugar• 5 carbons• 2’ C is missing an

oxygen• 3’ C and 5’ C are most

important

Phosphate•Same as you have seen

before•1 phosphorus atom•4 oxygen atoms•Overall –ve charge•DNA is –ve!!!

A Closer Look...

Adenine Thymine

Guanine Cytosine

A Closer Look...

Adenine Thymine

Guanine Cytosine

PURINES

The AGgiesare PURe.

PYRAMIDINES

A Closer Look...• A purine will always bond with a pyramidine• How nucleotide bases bond together:

• A & T share 2 H-bonds C & G share 3 H-bonds• **The H-bonding gives DNA its stability!

A Closer Look...*** Thymine can not bond with Guanine due to

lack of H-bonding***

*** Adenine can not bond with Cytosine due to lack of H-bonding***

Antiparallel...• DNA is said to run ANTI-PARALLEL – One strand runs in the 5’ 3’ direction– The other runs in the 3’ 5’ direction

• The 5’ refers to the 5th carbon on the deoxyribose sugar

• The 3’ refers to the 3rd carbon on the deoxyribose sugar

– The 5’ end terminates with an phosphate group

– The 3’ end terminates with an -OH group

Antiparallel...

DNA REPLICATIONThe Basics:• Cells can reproduce (as you have seen in mitosis)– Genetic information is divided equally from parent cell into

daughter cells

• Identical genetic information from parent to daughter cell is important to maintain identical cellular function

• From their structure, Watson and Crick could tell:– H-bonds between nucleotides could break– DNA could ‘unzip’– Each strand could act as a template to build a complementary

strand

DNA REPLICATIONMeselson & Stahl – important experiment• In 1958, suggested that DNA replication is

SEMI-CONSERVATIVE– Each daughter cell receives one strand of parental DNA– Conservative = one daughter receives both strand

DNA REPLICATIONProcedure• Grew E. Coli in a nutrient rich medium in 15N isotope• Allowed to replicate 17 times– thus all DNA should contain 15N

• Next, bacteria with 15N were transferred to a medium of 14N– Now, 14N should be found in daughter DNA– One strand should be heavier than the other– As replication continues, more 14N should be found

• All samples were centrifuged to separate by density• Results....

DNA REPLICATIONResults• Tube A = DNA of cells before switching to 14N• Tube B = DNA after 1st replication of 15N in 14N sol’n– Both 14N and 15N

• Tube C = DNA after 2nd replication – Intermediate band (14N+15N) and a light band (14N only)

Tube A Tube B Tube C

DNA REPLICATIONConclusion• Original strands are still present after many

replications, thus semi-conversative– They must act as a template

• NOT conservative or we would see one heavy band and one light band

Tube A Tube B Tube C

DNA REPLICATIONThe Roster• Many enzymes are used in this process– DNA helicase– DNA gyrase– DNA polymerase III– DNA polymerase I– DNA ligase– RNA primase

• All have a very specific function in this process

*Proteins can’t exist without DNA, but DNA has no function without proteins*

DNA REPLICATIONThe Process1. DNA Helicase unwinds DNA by breaking H-bonds▫ Obviously, they are going to want to rebound (anneal)▫ Luckily, single-stranded binding proteins (SSBs) bind to exposed

DNA blocking H-bonding

2. DNA Gyrase helps the unwinding process by relieving any excess tension

•DNA can’t be unwound all at once as it is too big.•The length of DNA in 1 chromosome is 1cm▫ The diameter of a cell 0.00005cm!!

•To avoid this we need to replicate in regions

DNA REPLICATION

DNA REPLICATIONThe Process3. DNA replication proceeds in the direction of the

replication fork ▫ Replication bubbles occur when two replication forks are

close to one another

4. DNA polymerase III starts to build complementary strand

DNA REPLICATIONThe Process•Before DNA polymerase III can initiate a new strand

by itself It needs a PRIMER

5. RNA PRIMASE allows an RNA primer to be added ▫ RNA primer marks the start

of the initiation sequence

DNA REPLICATIONThe Process6. Elongation - DNA polymerase:▫ Synthesizes DNA from 5’ 3’ direction▫ Adds free nucleotides to the 3’ end

•Elongation occurs easily on the 3’ 5’ strand▫ LEADING STRAND

•The 5’ 3’ strandcreates short fragments▫ LAGGING STRAND

DNA REPLICATIONThe Process•RNA primers must be continually added to the

5’3’ parent strand to create lagging strand▫ This allows DNA polymerase III to build short fragments

called OKAZAKI FRAGMENTS

DNA REPLICATIONThe Process7. DNA polymerase I•Removes RNA primers•Replaces them with

appropriate nucleotides

8. DNA Ligase•Joins one Okazaki

fragment to another•Creates a phosphodiester

bond

DNA REPLICATIONThe Process9. When mistakes occur,

DNA polymerase III & DNA polymerase I act as exonucleases

•They can backtrack to remove the incorrect nucleotide and replace it with the correct one

•A form of proof-reading

DNA REPLICATIONRecap