DNA Structure & Replication

• give a short summary of DNA replication

  1)DNA helicaseunwinds DNA by breaking the hydrogen bonds between bases

  2)SSBscoat the single DNA strands to prevent them from snapping back together.

  3)Primasemakes a short RNA primer as a starting point for replication

  4) Free nucleotides are attracted to their complementary bases and are held in place with hydrogen bonds (this makes them activated nucleotides: they contain 3 phosphate groups)

  5)DNA polymerasecatalyses condensation reactions to form phosphodiester bonds.

  -> The nucleotides lose the two extra phosphate groups

  (THIS PROVIDES ENERGY FOR REACTIONS)

  6)RNAseHremoves the RNA primers by hydrolyzing the RNA and DNA polymerase fills in the gap left by RNase H.

  7)Ligase enzymejoins the okazaki fragments together into one continuous strand.

  8) The final product is two exact copies of the starting material
• What does a low rate of mutation allow?

  Variation and survival of species, however certain mutations can lead to predisposition of cancers
• what happens if an incorrect nucleotide is inserted?

  DNA polymerase has proofreading activity (exonuclease 3' to 5').

  - the proof reading spots the mistake and replaces the incorrect nucleotide with the correct one
• How many mistakes does DNA polymerase make?

  1 mistake for every million nucleotides it copies
• describe discontinuous replication


  - The leading strand is replicated continuously

  - The other strand (the lagging strand) is replicated discontinuously in short fragments (Okazaki fragments) which are joined by enzyme DNA ligase (using ATP)

  
• since DNA polymerase can only synthesise DNA in the 5' to 3' direction,

  how are the two chains replicated simultaneously?

  via discontinuous replication
• What happens to RNA primers after DNA synthesis?

  They are removed
• what does the enzyme primase do?


  it synthesises short RNA primers , about 10 bases, which bind to DNA and form a short a short double stranded DNA:RNA hybrid for DNA polymerase.

  
  

  - RNA primer has a free 3'oh.
• what is a primer?

  A primer is a short nucleic acid sequence that provides

  a starting point for DNA synthesis
• what does the enzyme SSB do?

  it coats the single-stranded DNA and prevents it from annealing back to the complementary strand.
• What is a replication fork?

  The unwound region of the DNA where replication takes place

  
• What does the origin of replication contain?

  Binding sites for proteins that initiate replication
• circular and linear DS DNA molecules replicate differently.

  TRUE/FALSE

  TRUE
• how many origins of replication are there in eukaryotes (linear genome)?


  several
• how many origins of replication are there in bacteria (circular genome)?

  one unique site
• Where does DNA replication start?

  In the origin of replication:

  a small region where the 2 parental strands are separated and replication proceeds in a bidirectional manner from here
• Describe the main proteins involved in DNA replication and their functions.

  
• DNA polymerase has proof reading activity (exonuclease 3' to 5')

  TRUE/FALSE

  TRUE
• DNA polymerase cannot start a new strand itself but can extend the

  strand

  TRUE/FALSE

  TRUE
• Numerous proteins are needed before DNA polymerase can copy

  TRUE/FALSE

  TRUE
• in what direction does DNA polymerase synthesis DNA?

  5'-3' direction
• what do we mean by 'DNA COULD BE USED TO MAKE DNA'?

  enzyme DNA polymerase can use DNA as a template to make exact copies if provided with the correct ingredients.
• what happens when the DNA template is passed through the proteins of the replication factory?

  it is denatured and copied, one nucleotide at a time, using specialised proteins and enzymes.
• what is the replication factory?

  the cluster of proteins involved in DNA replication in the nucleus of a cell .
• where does DNA replication occur?

  in specialised regions within the nucleus of the cell

  (or within the cytoplasm in prokaryotes)
• when does DNA replication occur in the cell cycle?

  In eukaryotes, DNA replication takes place during the S phase of the cell cycle and can take 8-9 hours
• why is DNA replication semi-conservative?

  both "daughter" strands contain one of the original "parental" strands
• what are the steps of DNA replication?

  1. Helicase unwinds the double helix to expose two single DNA strands and create two replication forks.

  2. DNA replication takes place simultaneously at each fork. The mechanism of replication is identical at each fork.

  3. SSBs coat the single DNA strands to prevent them from snapping back together. (SSBs are easily displaced by DNA polymerase)

  4. Primase synthesises a short RNA primer complementary to a region of the DNA to be copied.

  5. DNA polymerase synthesises a new DNA strand by extending the RNA primer in the 5' to 3' direction.

  6. Both template strands have one DNA polymerase and move through the replication factory in the same direction.

  7. As DNA polymerase makes the strands discontinuously.

  8. RNAseH removes the RNA primers by hydrolyzing the RNA and DNA polymerase fills in the gap left by RNase H.

  9. Ligase enzyme joins the okazaki fragments together into one continuous strand
• what is needed to replicate a double stranded DNA?

  many proteins and enzymes are required which act in a co-ordinated and co-operative manner.
• what are the two ways to transfer genetic material?

  1) Inheritance (via copied DNA): REPLICATION

  2) Expression (via RNA): TRANSCRIPTION and TRANSLATION
• How is DNA arranged in prokaryotes?

  - Prokaryotes typically contain a single DS, supercoiled,

  circular chromosome.

  - Prokarytoic DNA is complexed with proteins to form

  nucleoids.

  - Bacteria also carry small circular extra-chromosomal

  molecules called plasmids
• How is DNA arranged in eukaryotes?

  - Eukaryotic chromosomes are long linear molecules of dsDNA, bonded to proteins to form chromatin.

  - Eukaryotes have closed circular DNA molecules in

  their mitochondria
• what are genomes divided into?

  discrete units called "genes
• are genomes DNA or RNA?

  genomes can be DNA or RNA
• what is a genome?

  The entire set of genes in the chromosomes of an organism, which carry all the information to code for that organism in the form of a sequence contained in the bases
• What does the spatial relationship between the 2 DNA strands create?

  A major (wide) and minor (narrow) groove

  -> the edges of the bases are accessible from these grooves
• what does it mean when we say that the chains of DNA are in an anti-parallel manner?

  the 5'end of one strand is paired with the 3'end of the other.
• what type of helix does DNA form?

  a right handed helix, with 10 nucleotides per turn, 1 turn is 3.4nm, and the helix diameter is 2nm
• How is DNA arranged?

  Normally double stranded (DS) with 2 chains coiled around each other and around a central axis in an anti-parallel manner.

  The hydrophobic bases face the inside whilst the phosphate faces the outside
• double stranded DNA has a higher relative absorbance at 260nm than single stranded DNA

  
  

  TRUE/FALSE

  FALSE

  
  

  single stranded DNA has a higher relative absorbance at 260nm than double stranded DNA