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									6 DNA Replication and Repair




            Topic 6            1
             DNA Replication
DNA lacks the ability to perform the process of
duplication alone
The machinery of the cell is required
DNA strands are complementary
  Each contains the information to replicate the
  alternate strand




                       Topic 6                     2
            DNA Replication
Semiconservative Replication
  Each daughter duplex is composed of one parent
  strand and one which is newly synthesized




                      Topic 6                      3
DNA Replication




      Topic 6     4
              DNA Replication
The machinery of replication
  Replication forks
    Points at which each of the replicated segments come
    together
    Each is a site where
        Parental double helix strands are separating
        Nucleotide incorporated into new complementary strands




                            Topic 6                              5
                  DNA Replication
The machinery of replication
  Some Enzyme Classes
    DNA helicase – unwinds DNA
    strands
       Single strands are stabilized by single
       strand DNA binding proteins
    DNA topoisomerase – relieves
    mechanical strain induced by
    unwinding
    DNA polymerases – synthesize
    new strands
    DNA ligase – forms new bonds
    between adjacent nucleotides

                                 Topic 6         6
             DNA Replication
The machinery of replication
  Properties of DNA polymerases
    Cannot initiate the formation of a new DNA strand
    Add nucleotides to the 3’ hydroxyl terminus
    A strand is required to provide the 3’OH
       Termed a primer
    All DNA polymerases have two requirements
       A template DNA strand to copy
       A primer strand to which nucleotides can be added
    All add nucleotides from the 5’ to 3’ direction



                           Topic 6                         7
             DNA Replication
The machinery of replication
  Semidiscontinuous replication
    OH group at the 3’ end of the primer reacts with the 5’ a-
    phosphate of the incoming nucleoside phosphate
    Polymerase molecules on both strands move in the 5’ to
    3’ direction
    One strand grows toward the replication fork
    The other grows away from the fork




                         Topic 6                          8
             DNA Replication
The machinery of replication
  Semidiscontinuous replication
    Strand growing towards the fork
       Continuous additions of nucleotides to 3’ end
    Strand growing away from fork
       Synthesized discontinuously
       As fragments
       Before synthesis the fork must move away
       Once initiated the fragment grows 5’ to 3’
       Subsequently each fragment is linked to the next
    The two daughter strands are synthesized by very
    different processes


                            Topic 6                       9
             DNA Replication
The machinery of replication
  Semidiscontinuous replication
    Strand synthesized continuously – leading strand
    Strand synthesized discontinuously – lagging strand

    Okazaki fragments
       Linked by DNA ligase




                         Topic 6                          10
             DNA Replication
The machinery of replication
  Semidiscontinuous replication
    Initiation not via DNA polymerase – by an RNA
    polymerase – a primase
       Constructs a short primer of RNA not DNA
       Required for both strands
    RNA primers subsequently removed
       Gaps filled with DNA
       Sealed by DNA ligase




                          Topic 6                   11
           DNA Replication
The machinery of replication




                     Topic 6   12
             DNA Replication
The machinery of replication
  Semidiscontinuous replication
    DNA polymerase occasionally inserts an incorrect
    nucleotide
    DNA polymerase has multiple enzymatic sites – an
    exonuclease site
    If an incorrect nucleotide is incorporated
       Strand tends to bulge
       Form a single-stranded 3’ terminus
       Enters the exonuclease site
       The polymerase stalls allowing the slow-acting exonuclease to
       excise the incorrect nucleotide

                            Topic 6                              13
              DNA Replication
The machinery of replication
  Additional features of eurkaryotic cells
    Incorporate nucleotides into DNA at slower rates
    Genome replicated in small portions
        Replicons
        50-300 base pairs
    Replicons close together tend to replicate simultaneously
    Timing of replication determined by
        Activity of the genes
        State of compaction




                                Topic 6                   14
              DNA Replication
The machinery of replication
  Additional features of eurkaryotic cells
    Yeast used as a model for eukaryotes
        Isolation of sequences which promote replication – autonomous
        replicating sequences (ARSs)
             Core element – 11 base pairs
             Binding site for protein complex – origin recognition
             complex (ORC)
    Many sites where DNA replication may be initiated
        Most inhibited by
           Nucleosome positioning
           Higher order chromatin structure


                            Topic 6                             15
              DNA Replication
The machinery of replication
  Additional features of eurkaryotic cells
    One replication per cycle – control
        The origin of replication – passage through a series of steps
            Origin of replication bound by ORC
            Licensing factors bind assemble the prereplication complex
            Licensing factors – at least six – Mcm2-Mcm7
            Mcm proteins move with the replication fork
            Mcm proteins are then displaced from DNA but remain in
            nucleus
            Mcm proteins cannot reassociate with an origin of
            replication which has already ‘fired’


                            Topic 6                              16
              DNA Replication
The machinery of replication
  Additional features of eurkaryotic cells
    Chromatin structure
        Nucleosomes and the replication fork
           Histones – H3H4 tetramers remain intact and are distributed
           between the daughter duplexes
           Old and new H3H4 tetramers found on each duplex
           H2A/H2B dimers – separate and bind randomly to H3H4
           tetramers already in place




                            Topic 6                              17
                    DNA Repair
DNA is susceptible to damage
  Ionizing radiation – breaks the backbone of the structure
  Metabolites – alter base structure
  UV Radiation – adjacent pyrimidines dimerise
  Mutations affect germ cells
     modified trait passed on
  Mutations affect somatic cells
     malignant transformation
     aging




                                Topic 6                       18
               DNA Repair
DNA is susceptible to damage
  Huge variety of repair mechanisms to repair DNA
  Proteins patrol DNA searching for alterations and
  distortions
  Most repair systems – excise the damaged section
  DNA duplex – each strand contains the information
  required for constructing its partner




                      Topic 6                   19
                DNA Repair
Nucleotide Excision Repair (NER)
  A ‘cut and patch’ mechanism
  Removes bulky lesions
    Pyrimidine dimers
    Chemical groups attached
  Two pathways
    Transcription coupled pathway
    Global pathway




                        Topic 6     20
                    DNA Repair
Base Excision Repair (BER)
  Initiated by a DNA glycosylase
     Recognizes alteration
     Removes base by cleavage of glycosidic bond between the base and
     deoxyribose
  Several specific types of DNA glycosylase – for specific
  modifications
     Uracil formed from hydrolytic removal of amino group of cytosine
     Formation of 8-hydroxyguanine – by oxygen free radicals
     Formation of 3-methyl adenine
  Following removal of the base – remaining deoxyribose
  phosphate is removed by endonuclease / phosphodiesterase
  Gap filled via DNA polymerase and sealed by DNA ligase


                             Topic 6                              21
             DNA Repair
Base Excision Repair (BER)




                   Topic 6   22
                   DNA Repair
Mismatch Repair
  Mismatch causes
  distortion of helix
     Recognized by a repair
     enzyme
     Repair system recognizes
     newly synthesized strand
     New strand recognized
     by presence of breaks




                           Topic 6   23
                  DNA Repair
Postreplication Repair
  Blockage of DNA
  polymerase progression
  by pyrimidine dimers
  and other lesions
  Replication can be
  restarted by synthesis of
  an Okazaki fragment




                          Topic 6   24

								
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