Rod Devenish; Week 3 MED1011; Biochemistry
DNA replication is semiconservative, replicated helices contain one parent strand and one replicated strand. DNA polymerase catalyses additions of amino acids to the 3' end of the growing strand. Nucleotides are added by complementary base pairing. The substrates, deoxyribonucleotide triphosphates are hydrolysed as added, releasing energy for DNA synthesis.
The DNA replication complex is in a fixed place and DNA is threaded through it for replication. The template strand interacts with a large protein complex called the replication complex (catalyses all the reactions). Circular chromosomes of prokaryotes have a single origin of replication, there are many in eukaryotes, and can occur at many sites simultaneously. The two circles formed by circular replication are split by topoisomerase. Topoisomerase is not needed in eukaryotes.
Localised unwinding of DNA takes place at the origin of replication by DNA helicase. Single strand binding proteins bind to the unwound strands to keep them apart. DNA polymerase looks like a hand, is a large complex with a groove for DNA to slide through. 'Finger' regions have precise shapes that recognise shapes of nucleotide bases.
DNA polymerase requires a primer, RNA primase catalyses short RNA primers. It does this before binding to DNA polymerase. DNA polymerase adds in the 5' to 3' direction on the leading strand, same on the lagging strand but Okazaki fragments (small fragments of DNA) are added to RNA primers, polymerase must 'skip' past the 5 end of one fragment to make the next. DNA polymerase I fills in nucleotides and removes primer, DNA ligase fills in gaps between fragments.
The newly replicated DNA strand is stabilised by a sliding DNA clamp which clamps around polymerase and DNA, increases efficacy of DNA replication.
At the end of DNA a primer is added, which is removed and the resultant free single stranded end is cut off, after each replication DNA shortens. Telomeres are added which are useless sequences to prevent this from damaging the genome. Telomerase enzyme is present in stem cells to prevent telomere exhaustion.
DNA repair is done through proofreading (errors occur every 10^5 base pairings) during replication, mismatch repair shortly after replication, excision repair over the life of the cell. Repair mechanisms lower incidence to 10^10. DNA polymerase I fills the gap from excision.
ddNTPs lack a hydroxyl group at the 3' end so no more nucleotides can be added and synthesis stops. Many strands of DNA are mixed with primers, DNA polymerase, normal dNTPs and ddNTPs. By using different colours for each base and looking at where colours are placed in endings you can sequence DNA. These differences in size are seen through electrophoresis. High throughput screening can sequence 50 million bases in 7 hours. Involves cutting DNA to 300-700bp segments, PCR amplifying each and attaching to microscopic beads. Beads are then used in sequencing reactions with flourescent nucleotides and a laser scanner reads addition of new base.