DNA replication accuracy Balance between accuracy and mutation (to allow evolution) About 1 mistake per basepairs Achieved by accuracy of DNA replication plus several editing and repair systems DNA is the only molecule that does this

DNA replication - basic rules DNA replication is semi-conservative Begins at specific origins and is bi-directional Requires DNA polymerase enzymes and other proteins Requires RNA primers Is semi-discontinuous

Semi-conservative replication Meselson-Stahl experiment (1958) Label E.coli DNA with 15 N by adding to growth medium Transfer to 14 N-containing medium Analyse density of DNA by centrifugation

Replication origins and direction Cairns: incubate E.coli in 3 H-thymidine Analyse replicating DNA by autoradiography Theta ( structures: DNA does not unwind completely By using different radio-isotopes at different times showed that replication is bi-directional Replication origins are specific AT-rich sequences Small circular chromosomes - single origin Long linear chromosomes - multiple origins

DNA polymerases and other proteins Specific proteins at origin to initiate replication: unwind DNA and keep it unwound Primase makes RNA primers Gyrase removes supercoils as DNA unwinds Several DNA polymerases (I, II, and III in E.coli) Can synthesise DNA in vitro using dATP, dTTP, dCTP and dGTP and some DNA as template

RNA primes DNA replication In vitro, DNA with this structure can act as template: In vivo, DNA replication is primed by a short bit of RNA: RNA primers are then degraded and gaps filled in 5353 primernew DNA 5353 RNA primernew DNA

Replication is semi-discontinuous DNA polymerases only catalyse 5 3 synthesis, so what happens at the replication fork? One strand (the lagging strand) is made in bits of a few 100bps (Okazaki fragments) which are then joined together by another enzyme - DNA ligase 5353 leading lagging