7.1 DNA'Structure'and'Replication'
DNA'Structure'
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DNA was crystallised and then targeted with an X-ray (whose beam became diffracted by DNA crystals)
The scattering pattern created by the diffracted X-ray was recorded on film
This pattern was then analysed to elucidate the structure of DNA
DNA is wrapped around histone proteins to form nucleosomes
Nucleosomes are grouped together (chromatosomes) and then arranged into fibres (chromatin)
DNA is usually organised as chromatin within the nucleus, except during cell division (when the chromatin
condenses to form chromosomes)
Euchromatin is more loosely packed and corresponds to active segments of DNA (i.e. active genes)
Heterochromatin is more densely packaged and corresponds to inactive segments of DNA
Different cells have different segments of DNA packaged as euchromatin and heterochromatin
A nucleosome consists of DNA and histone proteins
• DNA is wrapped around an octamer of histone proteins
• Nucleosomes are linked by an interconnecting H1 histone
Nucleosomes serve two key functions:
• They help to supercoil DNA (improves packaging)
• They help to regulate transcription
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Satellite DNA (e.g. short tandem repeats)
Telomeres (the terminal sections of chromosomes)
Introns (non-coding sequences within genes)
Non-coding genes (e.g. genes for tRNA or rRNA)
Gene regulatory sequences (e.g. promoters, enhancers, silencers)
Short tandem repeats (STRs) are short repeating segments within satellite DNA
The number of repeats for a particular loci will differ between individuals
The STRs can be excised and separated on a gel to create a distinct DNA profile of a given individual
(the more STR loci included in the profile, the more unique the DNA profile will be for the individual)
Hershey and Chase demonstrated that DNA was the genetic material by using radioactively labelled viruses
• Viruses were prepared with radioactive phosphorus (labels DNA) or radioactive sulphur (labels protein)
• Viruses then infected bacteria, before the bacteria and virus were separated via centrifugation
(bacteria is heavier and forms a pellet, while the smaller virus remains in the supernatant)
• When radioactive sulphur was used, radioactivity was detected in supernatant (not transferred to bacteria)
• When radioactive phosphorus was used, radioactivity was detected in pellet (WAS transferred to bacteria)
DNA'Replication'
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Replication occurs in a 5’ - 3’ direction (on the newly synthesised strand)
Unwinds and separates double stranded DNA (breaks H bonds between the base pairs)
Relieves torsional strain created by helicase action (i.e. prevents supercoiling)
Prevents DNA strands from re-annealing (SSB proteins will be displaced by DNA pol III)
Lays down a short RNA primer to provide an initiation point for polymerisation
(DNA pol III can only add nucleotides to the 3’-end of an existing nucleotide chain)
Extends the nucleotide chain from the primer
(dNTPs align opposite complementary bases and DNA pol III covalently joins them together)
Removes and replaces RNA primers with DNA nucleotides
Covalently joins Okazaki fragments together (on lagging strand)
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DNA strands are antiparallel, so DNA pol III moves in opposite directions on the two strands
On the leading strand, DNA pol III moves in the same direction as helicase - so synthesis is continuous
On the lagging strand, DNA pol III moves in the opposite direction to helicase – synthesis is discontinuous
The fragments generated on the lagging strand are called Okazaki fragments
Deoxynucleoside triphosphates (dNTPs) align opposite their complementary base partner
DNA pol III cleaves two of the phosphates and uses the energy to form a covalent phosphodiester bond
In this way, DNA pol III synthesises a new DNA strand
Dideoxynucleotides (ddNTPs) lack the 3’-hydroxyl group needed to form a phosphodiester bond
This means the inclusion of a ddNTP will terminate the extension of a DNA sequence at that point
Four PCR cycles are set up, each with a different ddNTP (ddA, ddT, ddG or ddC) and a stock of normal bases
Each time the ddNTP is incorporated the sequence stops, generating fragments
When these fragments are separated and then ordered according to length, the DNA sequence is discerned
This process can be automated by using fluorescently labelled ddNTPs that can be detected by machine
