DNA Replication

DNA replication...  The complementary base pairing provides a mechanism that allows for exact copying of the DNA molecule, time after time  At a simplistic level:  DNA molecule unwinds and unzips  hydrogen bonds joining the base pairs together break  leaves strands of DNA with exposed bases  exposed bases act as a template against which a new molecule is synthesised  also requires  DNA polymerase  supply of activated nucleotides (activated by the addition of two phosphate groups)

DNA replication...  Complementary bases align themselves opposite the exposed bases  A with T  G with C  DNA polymerase links  deoxyribose sugar  phosphate group of adjacent nucleotides together Annotate your diagrams to indicate the key features of DNA replication

DNA molecule consists of  two polynucleotide chains running in opposite directions  sugar phosphate backbone  bases sticking out  Complementary base pairing  purine with pyrimidine  AT and GC

DNA molecule  unwinds and unzips  hydrogen bonds holding the base pairs together break  exposes the bases of the polynucleotide chains  each chain can act as a template against which a new polynucleotide can be synthesised

 exposed DNA polynucleotide chains act as template against which new polynucleotide is synthesised  complementary bases align themselves opposite exposed bases of the polynucleotide  DNA polymerase bonds adjacent nucleotides together

 Two identical DNA molecules produced  Each consists of  one original polynucleotide strand  one newly-synthesised strand  Ensures consistency of instructions from one cell generation to the next

DNA replication – Meselson and Stahl  As described, DNA replication is semi-conservative i.e. in the new DNA molecule  one strand is original  other strand is newly-synthesised  However, in the 1950s scientists did not know exactly how replication took place  three possible ways were suggested:  conservative  semi-conservative  dispersive

Conservative  One completely new DNA molecule (double helix) is made  original molecule is conserved Semi-conservative  Each new DNA molecule contains  one original strand  one newly synthesised strand Dispersive  Each new DNA molecule is made from  bits of the old DNA  bits of newly- synthesised DNA Replication options…

Meselson and Stahl experiments...  Semi-conservative replication is the most obvious choice  Confirmed by Meselson and Stahl in 1958  Grew the bacterium Escherichia coli in a medium that contained the heavy isotope of nitrogen  nitrogen 15 or 15 N  heavy isotope was incorporated into structures containing nitrogen such as DNA  if grown in this medium for long enough all DNA contained the heavy nitrogen

... Meselson and Stahl experiments  E. coli was then switched to a new medium  contained nitrogen 14 or 14 N lighter isotope than 15 N  any new DNA produced contained the lighter isotope  They were left in the medium long enough to replicate one or more times  about 45 minutes for one replication  DNA was then extracted  centrifuged in caesium chloride  DNA was spun out  in bands depending upon its density

Grown in 15 N  All of the DNA contains the heavy isotope  it is heavy  comes to rest at the bottom of the centrifuge tube Grown in 14 N (G1)  The DNA contains two strands of DNA  one is the original heavy  other is newly synthesised and contains 14 N Grown in 14 N (G2)  There are two bands of DNA  one is a mixture of heavy and light  other contains only 14 N Grown in 14 N (G3)  There are two bands of DNA  one is a mixture of heavy and light  other contains only 14 N Results of Meselson and Stahl experiments…