Lecture 22

• Basic theme in DNA replication 1

  The two strands of the double helix have a great affinity for one another. strands are intertwined, and held together by H bonding between base pairs

  double helix is stabilized by aromatic stacking of successive bases
• basic theme in DNA replication 2

  two strands must be unwound and completely separated

  accomplished by energy dependent enzyme systems
• basic theme in DNA replication 3

  Replication is highly accurate

  multiple proof reading and editing mechanisms that increase the accuracy of DNA synthesis

  must be accurate because, unlike transcription and translation, it constitutes the genetic heritage of future organisms
• basic theme DNA replication 5

  Specialized enzyme systems are used to synthesize structures called telomeres at the ends of the chromosome
• DNA double helix

  tightly packed structure in which the base pairs are relatively inaccessible to enzyme systems that catalyze replication

  The space filling model shows just how tightly the DNA is packed
• DNA template

  copied and directs the synthesis of the complementary DNA sequence
• primer

  initial sequence of DNA at the 5’ end of the new DNA. The primer provides a 3' hydroxyl that attacks the incoming nucleotide. (RNA synthesis does not require a primer.)
• deoxynucleotide triphosphates

  four

  provide the nucleoside monophosphate unit that is added to the growing chain of DNA
• DNA pol

  complex of enzymes that catalyze the process of DNA elongation
• Metal ions

  such as Mg++ and several other protein factors are also required
• In addition to the core requirements for DNA replication, other factors are required to open up the parental double helix and to accommodate the special needs of lagging strand replication
• Requirements for DNA replication - additional

  ADDITIONAL

  • SS binding proteins (SSB)
  • helicase
  • topoisomerase
  • primase
  • nucleotide triphosphates
  • ligase
• SSBs

  help keep the strands apart after they are separated
• helicase

  enzyme catalyzes energy dependent strand separation
• topoisomerases

  collectively relax the supercoiled DNA that is created by the action of the helicase
• primase

  creates an RNA primer region in lagging strand fragments

  provides starting material so DNA polymerase III can add segments of DNA
• nucleotide triphosphates (NTPs)

  provide energy for the action of helicase and topoisomerase
• DNA polymerase 1

  removes the RNA primer nucleotides from the lagging strand segments and replaces them with the appropriate deoxynucleotides
• ligase

  closes the gaps between segments of lagging strand DNA
• DNA synthesis step 1

  occurs on template strand of DNA

  new DNA added onto 3' end of primer, synthesis is 5' to 3'

  primer and newly synthesized material run in anti-parallel direction to template strand
• DNA synthesis step 2

  dTTP will diffuse to occupy open site and H bond to A residue on template (TA base pair)

  3' OH group of primer will launch a NU attack on P atom of alpha-P of dTTNP
• DNA synthesis step 3

  3' OH group of primer forms phosphodiester linkage with alpha-P residue and releases inorganic pyrophosphate, hydrolyzed to 2 molecules of Pi, pulls reaction in direction of synthesis

  DNA copy now one nt. longer, ready for another round

  now empty binding site for dATP to T residue
• DNA synthesis step 4

  dATP occupy open site, H bond to T

  3' OH group NU attack on P atom of alpha-P of dATP
• DNA synthesis step 5

  same as 3

  now empty binding site for dGTP for C residue

  process of DNA elongation will continue as long as there are open positions on template and all other reaction components are in adequate amounts
• strand separation problem

  tightly wound parental strands of duplex DNA must be separated so that primer strands can bind, and the DNA polymerase reaction can occur
• ATP-dependent helicase separates DNA strand

  Spontaneous strand separation is too slow to permit the efficient replication of DNA. Rapid strand separation is achieved by an ATP-dependent helicase enzyme
• Helicase mechanism

  separation of DNA strand by ATP dependent helicase

  unwind ssDNA

  A1 and B1 have a cleft that closes when ATP is bound

  when ATP is hydrolyzed, the cleft opens, pulling the DNA from domain B1 toward A1
• consequences of helicase activity

  additional strain is introduced into the DNA molecule, causing it to be overwound in surrounding regions
• Formation of supercoiled structures

  referred to as topoisomers

  If nothing were done to relieve the supercoiling of adjacent DNA, the activity of helicase would be inhibited and strand separation would cease
• topoisomerases function in supercoiled structures


  first increase and then to eventually relieve the strain of supercoiled DNA
• topoisomerase 2 (DNA gyrase) - supercoiled structures

  catalyzes an ATP-driven formation of negative supercoils, making the DNA more susceptible to unwinding by helicase
• topoisomerase 1 - supercoiled structures

  cleaves one DNA strand of supercoiled DNA, rotates in a controlled fashion around the other strand, and then religates the cleaved strand. results in the partial or complete relaxation of supercoiled DNA
• topoisomerase 1 mechanism

  cleaves one strand of supercoiled DNA

  rotates 360 degrees around the intact strand of DNA, religates the cleaved DNA strand

  process occurs repeatedly, resulting in the relaxation of the supercoiled DNA
• semiconservative replication

  That is, during replication, the parental strands are separated, and each parental strand pairs with one newly synthesized strand
• properties of replication

  two strands of DNA duplex run in opposite directions

  semiconservative model

  new strands are antiparallel and complementary to the parental DNA strands
• directionality of replication

  proceeds from a set point and both strands copied simultaneously

  DNA polymerase reaction only runs in 5' to 3'

  one strand could be synthesized in a continuous manner from 5' to 3', but it was initially unclear how the other strand could be synthesized in the 3' to 5' direction
• It has been confirmed that the DNA polymerase can add on to a 3’ of a nucleotide strand, synthesizing the new DNA from the 5’ to 3’ end
• leading strand

  s synthesized in the 5’--> 3’ direction, which is in the same direction as the mechanism of DNA polymerase
• lagging strand


  synthesized by constructing a series of short segments in the 5’--> 3’ direction and then joining the segments together.

  net flow of synthesis is 3' to 5'
• DNA replication proceeds in the unwound area on both strands simultaneously, and the net flow of DNA synthesis is in the same direction