7.1 DNA STRUCTURE AND REPLICATION

• The sugar-phosphate backbone is on the outside bases are on the inside
• The strand is held together by Hydrogen bonds between the bases
• Complementary Base Pairing: A only pairs with T. C only pairs with G
• Anti-parallel strand: Two strands run in opposite directions 
• DNA twists into a double helix through the use of Hydrogen bonding
• Covalent bond: Link between 5' and 3' ends of carbon molecules

Prokaryotic DNA: Naked circular DNA and plasmids [not associated with Histone proteins]

Eukaryotic DNA: DNA is associated with Histone proteins

Nucleosome: Length of DNA of approcimately 150 base pairs wrapped around a core of 8 Histone proteins [two pairs of four]

Nucleosomes are formed by wrapped DNA around Histone proteins

Eukaryotic DNA is supercoiled to:

•  Be able to pack genetic material into the nucleus
• Organize DNA to allow cell division to occur
• To control DNA expression
• Allow cells to specialize
• Transcription of chromatin can be promoted or inhibited

Nucleosomes helps supercoil the DNA

DNA is kept in a double helix by:

• phospo-diester bonds of the sugar-phosphate backbone
• hydrogen bonds between the complementary bases

DNA Replication in Prokaryotes can only be initiated at one point

DNA Replication in Eukaryotes can be initiated at many points

STANDARD LEVEL

1. DNA Helicase unwinds the and unzips DNA

The two strands are seperated by breaking Hydrogen bonds between complementary base pairs. The two separated strands become Template strands

2. DNA Polymerase creates complementary strands

DNA Polymerase always moves in a 5' to 3' direction. It catalyses the phopodisester bonds between sugar and phosphate molecules. 

HIGHER LEVEL

RNA Primers provide an attachment and initiation point for DNA Polymerase iii

RNA Primers: Short sequence of RNA nucleotides

DNA Replication moves in a 5' to 3' direction. The 5' is the end to which a new nucleotide attaches

Leading Strand: DNA is being replicated continously in a 5' to 3' direction

Lagging Strand: DNA is being replicated discontinously in small fragments in the 5' to 3' direction away from the replication fork

DNA Polymerase iii adds free nucleotides to C3. The strand grows from the 3' end

New strand: 5' to 3' direction 

Template strand 3' to 5' direction

Two new strands which are identical to the Template strand

Semi-Conservative: New strand contains one new and one template strand

1. DNA Gyrase: Relieves strain and prevents supercoiling of DNA double helix

2. DNA Helicase: Unwinds and seperates double stranded DNA by breaking Hydrogen bonds between Complementary bases

3. RNA Primase: Synthesizes a short RNA Primer on each template strand to provide an attachment and initiation point for DNA Polymerase iii

4. DNA Polymerase iii: Adds nucleotides to the 3' end of the polynucleotide chain, synthesizing in a 5' to 3' direction

5. DNA Polymerase i: Removes the RNA Primers and replaces them with DNA

6. DNA Ligase: Joins the Okazaki fragments together to create a continuous strand

DNA Replication occurs [during S phase] of Interphase in preparation for cell devision

Helicase unwinds the double helix seperating the strands of DNA

Helicase breaks the hydrogen bonds between the two strands 

Single stranded binding proteins keep the separated strands apart so that nucleotides can bind to the strands

DNA Gyrase moves in advance of Helicase and relieves strain and prevents the DNA supercoiling again

Each strand of parent DNA is used as a template for the synthesis of the new strands 

Synthesis always occurs in a 5' to 3' direction on each new strand

Synthesis is continuous on the leading strand and discontinuous on the lagging strand 

This leads to the formation of Okazaki fragments on the lagging stand

To synthesize a new strand first an RNA Primer is synthesized on the parent DNA using RNA Primase 

DNA Polymerase iii adds free nucleotides to the 3' end according to complementary base pairing rules 

Nucleotides are added

DNA Polymerase i removes the RNA primers and replaces them with DNA

DNA Ligase joins Okazaki fragments on the lagging strand 

Each new DNA molecule contains both a parent and a newly synthesized strand - DNA Replication is said to be Semi - Conservative

During Splicing: 

Introns are edited out of mRNA

mRNA is translated by Ribosomes into Polypeptides

Only Exons code for Polypeptides

Telomeres: Repetitve sequences at the ends of chromosomes that protect the ends of the chromosome

With every cell devision short stretches of DNA are lost from the Telomeres. Prevents at the ends of chromosomes from being lost each time DNA Replication occurs

Genes for tRNA and rRNA

Genes that code for RNA molecules that do not get transcribed instead fold and form tRNA molecules [important in Translation]. rRNA forms ribosomal RNA that forms the structure of the ribosome

Were proteins or chromosomes the genetic material of cells?

Alfred Hershey and Martha Chase found out if protein or DNA was the genetic material of viruses

• Viruses infect cells and transform them into virus-producing factories
• Viruses inject genetic material into the cell
• Non-genetic part of the virus remains outside the cell
• The infected cell produces many copies of the virus
• The cell bursts releasing the copied virus

Studied the T2 Bacteriophage which infects the E.Coli bacterium

The T2 Bacteriophage consists of:

Protein Coat

DNA inside the coat

1. Amino acids containing radioactive isotopes were used to label the virus

• Sulphur S35 for Protein coat
• Phosphorous P32 for DNA

2. Comined T2 Bacteriophage with E.Coli bateria

3. Centrifuge was used to seperate the bacterium and bacteriophage

Results:

• The smaller virus remained in the supernatant [liquid]
• The bacteria formed a pellet
• Sulphur S35 remained in the supernatant
• Phosphorous P32 was found in the pellet

Conclusion: 

DNA was the genetic material used by the viruses because DNA [P32] was being transferred into the bacteria

What is the structure of DNA [look like]?

Rosalind Franklin and Maurice Wilkins investigated the structure of DNA using X-Ray Diffraction

X-Ray Diffraction: When X-Rays are directed at a material some is scattered by the material. This scattering is known as diffraction

For X-Ray Diffraction to be successful:

• Material should be crystalized [crystals remain undistured while cooling - grow according to a regular pattern]

So that the repeating pattern causes diffraction to occur in a regular way

DNA cannot be crystalized but molecules arranged regularly enough for technique to work

Atoms, molecules or ions that make up solids can be arranged in an orderly, repeating pattern

How to figure out base sequence of a DNA strand?

Method used was based on the nucleotides of dideoxyribonucleic acid [ddna].

Dideoxyribose have no OH group on carbon atom 3

If a dideoxynucleotide is at the end of a strand of DNA: NO site to which another nucleotide can be added by a 5' to 3' linkage

1. In sequencing machine single-stranded copies of the DNA being sequenced are mixed with 

• DNA Polymerase
• Normal DNA nucleotides
• Small numbers of ddna nucleotides

Replication is repeated four times: Once with dideoxyribosenucleotides with each base  A T C G

Results: Fragments of DNA vary in length - depending on how far replication got before it was terminated because a ddNA nucleotide was added to the end of the chain

2. Fragments were seperated according to length by Gel Electrophoresis with four tracks

• One for each base in the ddNA nucleotide that terminated replication
• Each band in gel repersents one length of DNA fragment produced by replication
• All fragments of same length end in same base
  
• A band in one of the four tracks for each length of fragment

The whole base sequence can easily be deduced. Fluorescent markers allowed the base sequence to be read by a machine.

Dideoxyribosenucleic acid stops DNA Replication when it is added to a new DNA strand

Fluroscent dye markers are attached to dideoxyribosenucleic acids so that the base present when replication stops can be identified. The base on the parent strand can be deduced

New strands of different lengths are produced

The length of a strand and base are identified by sequencing machines

Tandem Repeats: Short sequences of non-coding DNA normally the length of 2 - 5 base pairs that are repeated numerous times

Adjacent sections of DNA that have the same base sequence

Variable number Tandem Repeats - Number of repeats varies between different individuals 

DNA Profiling is based on variable number of tandem repeats