DNA REPLICATION

  1. SEMICONSERVATIVE REPLICATION
    1. Conservative replication
      1. This model, which was shown to be incorrect, predicted that after replication, the parent double-stranded DNA would remain intact while the daughter double-stranded DNA would be entirely newly synthesized
    2. Semiconservative replication
      1. This model predicts that both daughter DNA molecules would contain one strand each from the parent DNA and one entirely newly synthesized strand
  2. Enzymology of DNA Replication
    1. DNA polymerases
      1. Substrates
        1. Needs all four deoxynucleoside 5'-triphosphates
          1. 5' mono- and di- phosphates and 3' mono-, di-, or tri-phosphates do not work
        2. Needs a DNA template
        3. Needs a nucleotide primer with free 3'-OH group
          1. All nucleic acids are synthesized in the 5' to 3' direction
      2. Reaction
        1. Poly(deoxynucleotide)n-3'-OH + dNTP ó Poly(deoxynucleotide)n+1-3'-OH + PPi
        2. Monophosphates will not work
          1. Formation of the phosphodiester bond is extremely endergonic
          2. Energy released from breaking pyrophosphate from NTP provides the energy for polymerization
      3. Prokaryotic DNA polymerases
        1. DNA polymerase I
          1. 3' to 5' polymerase activity
            1. Catalyze DNA polymerization
          2. 5' to 3' exonuclease activity
            1. Nucleotides are removed from 5'-P terminus
            2. Functions to remove ribonucleotide primers
          3. Functions to remove RNA primer and replace it with deoxyribonucleotides
        2. DNA polymerase II
          1. Used in DNA repair
          2. Has 5' - 3' polymerase activity and 3' - 5' exonuclease activity
        3. DNA polymerase III
          1. Major enzyme used in replication
          2. Has 5' - 3' polymerase activity and 3' - 5' exonuclease activity
      4. Proofreading
        1. If an incorrect base is added to the growing DNA chain DNA polymerases can back up and remove that base and then continue
          1. The removal of the incorrect base is referred to as 3' to 5' exonuclease activity
    2. DNA ligase
      1. Function
        1. Forms phosphodiester bonds between two segments of DNA
      2. Mechanism
        1. Joins 3'-OH to a 5'-monophosphate group
    3. DNA Gyrase
      1. Function
        1. Unwinds DNA helix into single-stranded DNA so that replication can proceed
    4. Primase / RNA polymerase
      1. Function
        1. DNA polymerase must connect nucleotides to 3'-OH group
          1. Cannot lay down first nucleotide
        2. RNA polymerase does not have this requirement
          1. A few ribonucleotides laid down by RNA polymerase can serve as a primer for DNA polymerase
      2. RNA polymerase
        1. Primes the leading (continuous strand)
      3. Primase
        1. Primes lagging (discontinuous strand)
      4. Primer
        1. 1 to 60 bases
        2. Provides 3'-OH group for DNA polymerase III to add a deoxynucleotide
  3. TOPOGRAPHY OF DNA REPLICATION
    1. Unwinding of parental DNA during replication causes stress in the unreplicated portion of DNA, which if not relieved, could prevent the replication fork from moving upstream
      1. Bacterial chromosomes cannot relieve their stress as it is a covalently closed circle
      2. Eukaryotic chromosomes, though linear, are too large to rotate to relieve stress
    2. DNA topoisomerases
      1. DNA gyrase, a topoisomerase, uses breaking, twisting, and ligating ability to remove stress
        1. DNA gyrase wraps DNA around it, cuts both strands of DNA. then passes DNA through the gap of broken strands and reforms the phosphodiester backbone
  4. replication fork
    1. Origin of replication
      1. DNA replication begins at specific regions of DNA referred to as 'Origins of Replication' or ori sites
        1. Prokaryotes contain only one ori site
        2. Eukaryotes contain multiple ori sites per chromosome
          1. Multiple ori sites are needed due to the larger size of DNA in eukaryotes and the slower speed of DNA replication of eukaryotic DNA polymerases
    2. Replication forks
      1. DNA is replicated bi-directionally from each ori site
      2. A replication fork is the area of DNA that is being unwound prior to replication
      3. There are two replication forks for every one ori
        1. As DNA replication begins continuously on one strand, the first Okazaki fragment produced becomes the leading strand for the other replication fork
    3. Advance of the replication fork and unwinding the helix
      1. Addition to nucleotides and unwinding of DNA are two different processes
        1. DNA polymerase III cannot unwind DNA
        2. Unwinding is catalyzed by enzymes called helicases
      2. Single-stranded binding proteins
        1. DNA polymerase III is not directly behind the helicase
          1. There is therefore some single-stranded DNA in the leading strand
          2. There is a larger gap of single stranded-DNA on the lagging strand
        2. Single-stranded binding proteins coats single-stranded DNA so they cannot reform hydrogen bonds
          1. These single-stranded binding proteins must be displaced by DNA polymerase III or another enzyme
  5. CONTINUOUS REPLICATION
    1. DNA helicase
      1. Unwinds DNA double helix
        1. Separates double-stranded DNA into single-stranded sections
        2. Starts at ori site
        3. Results in topographical stress
    2. Single-stranded DNA binding proteins
      1. Keeps complimentary strands of DNA from reannealing
    3. DNA topoisomerases (e.g., DNA gyrase)
      1. Relieves stress caused by helicases
    4. Primase (RNA polymerase)
      1. Lays down RNA primer
    5. DNA Polymerase III
      1. Adds nucleotides to 3' end of primer
      2. Adds nucleotides to 3' end of growing DNA polymer
    6. DNA Ligase
      1. Seals the ends of the newly created DNA circle
  6. Discontinuous replicatioN
    1. Replication fork
      1. At the replication fork, one strand is synthesized continuously, the other discontinuously, because the strands are antiparallel
        1. All DNA is synthesized in the 5' to 3' direction
    2. Steps
      1. DNA is unwound
      2. RNA primer is made at fork
      3. DNA polymerase adds nucleotides to 3' end
      4. DNA polymerase runs into previous primer
      5. Cycle starts over again (1 through 4)
      6. DNA polymerase I removes RNA primer and replaces it with deoxynucleotides
    3. Okazaki fragments
      1. Size
        1. Eukaryotes
          1. 100 - 200 bases
        2. Prokaryotes
          1. 1000 - 2000 bases
      2. Connecting Okazaki fragments
        1. Okazaki fragments are joined to form a continuous DNA containing no ribonucleotide
        2. DNA polymerase I
          1. Removes the primer ribonucleotides
          2. Replaces them with deoxyribonucleotides
        3. DNA ligase
          1. Catalyzes formation of phosphoester bond between nucleotides
        4. No 3' to 5' polymerase
          1. It would be simpler, however, evolution has not taken this course
  7. Eukaryotes
    1. Rate
      1. Eukaryotic DNA polymerases are slower than bacterial DNA polymerases
        1. Can replicate about 500 - 5,000 bases per minute
        2. Bacterial can replicate about 105 (100,000) per minute
      2. To make up for the slower replication, eukaryotes have more origins of replication
        1. Mammals have around 12,000 ori sites
    2. DNA polymerases
      1. DNA polymerase a
        1. Polymerizes the discontinuous strand
      2. DNA polymerase b
        1. Is used in DNA repair
      3. DNA polymerase d
        1. Polymerizes the continuous strand
      4. DNA polymerase g
        1. Found in mitochondria and chloroplasts