6 DNA Replication and Repair

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6 DNA Replication and Repair
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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

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DNA Replication

• Semiconservative Replication
• Each daughter duplex is composed of one parent
  strand and one which is newly synthesized

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DNA Replication
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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

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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

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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 3OH
• 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

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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

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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

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DNA Replication

• The machinery of replication
• Semidiscontinuous replication
• Strand synthesized continuously leading strand
• Strand synthesized discontinuously lagging
  strand
• Okazaki fragments
• Linked by DNA ligase

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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

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DNA Replication

• The machinery of replication

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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

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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

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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

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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

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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

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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

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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

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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

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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

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DNA Repair

• Base Excision Repair (BER)

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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

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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