DNA Replication

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DNA Replication

• AHMP 5406

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Objectives

• Outline the mechanisms of eukaryotic DNA
  replication
• Describe the cellular mechanisms that help avoid
  error generation during DNA synthesis
• Describe the possible pathways of DNA repair
• Relate chromatin density and the cell cycle to
  DNA replication

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DNA Replication

• The process of copying DS DNA by templated
  polymerization
• In Eukaryotes occurs only during S phase
• Overall replication scheme similar to
  prokaryotes

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DNA Replication

• Base pairing is responsible for DNA replication
  and repair
• Multiple initiation points
• Linear chromosome (Proks. circular)
• Many polymerases and accessory factors required

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Chromosome Size and Topology
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DNA replication is semi-conservative

• During one round of replication
• One strand used as template

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Repl. begins at specific chromosomal sites

• Replication origins
• Regardless of organism are
• unique DNA segments with multiple short repeats
• recognized by multimeric origin-binding proteins
• usually contain an A-T rich stretch

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Most DNA replication is bidirectional
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Eukaryotic Chromosome Replication

• DNA replication are very similar in proks and
  euks
• Differences
• Euks have many chromosomes
• one in prokaryotes
• The problem with nucleosomes
• euk DNA is packaged
• wrapped around histones
• In eukaryotes DNA and histones must be doubled
  with each cell division

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Eukaryotic Replication

• DNA synthesis
• In eukaryotes
• small portion of the cell cycle (S)
• continuously in prokaryotes
• Eukaryotes have more DNA to replicate
• How is this accomplished?
• Multiple origins of replication
• prokaryotes one origin OriC
• Two different polymerases

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Problems that must be overcome for DNA polymerase
to copy DNA

• DNA polymerases cant melt duplex DNA
• Must be separated for copying
• DNA polymerases can only elongate a preexisting
  DNA or RNA strand (the primer)
• Strands in the DNA duplex are opposite in
  chemical polarity
• All DNA polymerases catalyze nucleotide addition
  at 3?-hydroxyl end
• Strands can grow only in the 5? to 3? direction

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Structure of DNA Rep. Fork

• Both daughter strands polymerized in
• 5-3 direction
• Lagging strand DNA synth. in short segments
• Okazaki fragments

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Proteins at the fork form a replication machine

• Mammalian replication fork

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Specialized enzymes

• Helicases separate two parental DNA strands
• Polymerases synthesize primers and DNA
• Accessory proteins promote tight binding of
  enzymes to DNA
• Increase polymerase speed and efficiency (sliding
  clamp)
• Editing exonucleases work with polymerases
• Topoisomerases convert supercoiled DNA to the
  relaxed form

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DNA Helicase

• Hexameric ring
• Separate DNA strands
• Use ATP hydrolysis for Energy

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Primase

• Activated by helicase
• Synthesizes short RNA primer
• Uses DNA as template

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Sliding clamp

• Keeps DNA polymerases attached to DNA strand
• Assisted by clamp loader through ATP hydrolysis
• Will disassociate if DNA pol reaches DS DNA

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Single stranded binding proteins

• Bind tightly and
• cooperatively to SS DNA
• Do not cover bases
• Remain available for templating
• Aid in stabilizing unwound
• DNA
• Prevent hairpin structures

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Mammalian DNA polymerases

• Synthesize new DNA strand
• Requires primer
• DNA Pol a
• Associated with primase
• DNA Pol d
• Elongates

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Mammalian DNA Polymerases

• a Repair and Replication and primase function
• b Repair function
• g Mitochondrial DNA polymerase
• d Replication with PCNA (processivity factor)
• e Replication

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Topoisomerase

• Some proteins change topology of DNA
• Helicase can unwind the DNA duplex
• induce formation of supercoils
• Topoisomerases catalyze addition or removal of
  supercoils

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Topoisomerase

• Type I topoisomerase relax DNA by nicking and
  closing one strand of duplex DNA
• Covalently attach to DNA phosphate
• Allow rotation

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Topoisomerase

• Type II topoisomerase change DNA topology by
  breaking and rejoining double stranded DNA

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Action of E coli Topoisomerase I
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Type II topoisomerases (gyrases) change DNA
topology by breaking and rejoining
double-stranded DNA
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Replicated circular DNA molecules are separated
by type II topoisomerases
Linear daughter chromatids also are separated by
type II topoisomerases
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The eukaryotic replication machinery is generally
similar to that of E. coli
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More on Telomeres
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Telomeres

• Further evidence of a relationship b/w telomere
  length and aging in humans
• Disorder called progerias (premature aging)
• Hutchinson-Gilford Syndrome (severe) death in
  the teen years
• Werner Syndrome (less severe) death usually in
  the 40s

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Telomere Replication

• Regions of DNA at each end of a linear chromosome
• Required for replication and stability of that
  chromosome.
• Human somatic cells (grown in culture) divide
  only a limited number of times (20-70
  generations)

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Telomere Replication

• Correlation between telomere length and the
  number of cell divisions preceding senescence and
  death
• Cells with longer telomeres survive longer (more
  divisions) than cells with short telomeres

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Problem with Telomeres

• DNA polymerase require free 3OH end
• cannot replace the RNA primer
• at the terminus of the lagging strand.
• If not remedied, the DNA would become shorter and
  shorter
• Telomerase resolves the terminal primer problem

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Telomerase

• Telomerase enzyme made up of both protein and
  RNA
• RNA component is base sequence complementary to
  telomere repeat unit
• Catalyzes synthesis of new DNA using RNA as
  template

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End-Replication Problem
5 3

5 3
Process Okazaki Fragments
5 3

5 3
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Telomere Structure
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G-rich
C-rich

• Telomeres composed of short (6-10 bp) repeats
• G-rich in one strand, C-rich in other

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Telomerase

• Germ-line cells possess telomerase activity
• Most human somatic cells lack telomerase activity
• Cultured immortal cell lines have been shown to
  have telomerase activity
• Possible cancer therapy may be to control
  telomerase activity in cancer cells