Replication

1
Replication
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Outline

• I. Dogma
• II. DNA structure (genes, chromosomes)-length
  of human genome
• III. Replication
• A. Initiation
• 1. Proteins involved
• 2. Enzymes involved
• 3. Sequence of events
• B. Elongation
• C. Termination
• D. Proofreading

• IV. Repair
• A. Mismatch (dam)
• B. Base Excision (single base)
• C. Nucleotide excision (DNA segment)
• V. Advanced Topics
• A. Recombination
• B. Transposons

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Central Dogma of Information Flow

• Historical overview of DNA as an informational
  molecule (?where did this dogma come from?)

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Historical Basis I

• Friedrich Miescher (1868) isolated acidic
  component from puss, now known to be DNA
• basic component protein
• Griffin (1928) DNA inserted into bacteria changed
  the behavior of bacteria

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Historical Basis II Avery, McLeod and McCarthy
(1949) Fig. 10-12(a-e) Lehninger POB 3rd Ed.
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Historical Basis IIIFig. 10-13 Lehninger POB 3rd
Ed.

• Hershey-Chase experiment
• Shows that the protein of a bacteriophage does
  not enter bacteria.

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Wagging the Dogma

• 1976 David Baltimore et al.
• RNA viruses such as HIV
• Pruisner prion hypothesis

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DNA Structure

• Last semester ds, base-paired, anti-parallel
  ?-helix which could be melted
• Now levels of structure
• 1o structure the sequence of nt
• 2o structure the ?-helix
• 3o structurechromosome
• supersecondary structures

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Prokaryotic DNA Packing and Wieses Stupid Hose
Trick
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Eukaryotic DNA Packing - Histones

• Lys/Arg rich protein
• H1, H2a, H2b, H3, H4
• Octamer forms two each not H1

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DNA Packing - The NucleosomeFig. 24-24(c)
Lehninger POB 3rd Ed.
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DNA Packing - Beads on a String Fig. 24-23(b)
Lehninger POB 3rd Ed.
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DNA Packing SolenoidLeft Fig. 24-27(a)
Lehninger POB 3rd Ed.Right Fig. 38-3 Harpers
ROB 24th Ed.
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DNA Packing - LoopingFrom ?
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DNA Packing - MinibandsFrom ?
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DNA Packing - ChromosomeFig. 24-7 Lehninger POB
3rd Ed.
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Not All DNA is Packed Equally

• DNase insensitive, DNase sensitive, DNase
  hypersensitive
• Euchromatin- and heterochromatin- (facultative
  and constitutive)
• Barr bodies-

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Replication

• Replication- the synthesis of DNA using itself as
  a template.
• Three stages Initiation, Elongation, Termination

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Replication is Semiconservative

• Meselson-Stahl Experiment
• Grow bacteria on 15N source- get bottom band
• Grow bacteria on 14N source- get top band
• Grow bacteria on 15N source, then switch to 14N -
  get band halfway in between

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Replication is 5 ? 3(Pulse-chase experiment)

• Pulse with radioactive tracer
• Chase with large mass unlabeled
• One end of molecule labeled

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Replication Begins at a Specific Place Each Time
The Origin of replication (ori)

• If starts at random If always starts same place

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The Origin

• Analogous to Fig 25-11 Lehninger POB 3rd Ed
• Not to scale

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Consensus Sequence
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Replication is Bidirectional(Usually)Fig. 25-3
Lehninger POB 3rd Ed.

• Theta structure

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Initiation
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Ori C (AKA Dna C) Binds the Origin

• 250 bp protected from DNase

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DNA is Melted

• Replication bubble

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ssb (single strand binding) Proteins Binds Around
ori C

• Prevents DNA from reannealing
• Prevents nuclease activity

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Helicase Unwinds DNA
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Primase Synthesizes an RNA Primer 10-200 nt Long
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Elongation
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There Are Multiple DNA Polymerases, With
Different Functions

• Prokaryote
• I- fill in RNA gap, repair
• II- ?
• III- elongation

• Eukaryote (19 IDd)
• ?- elongation
• ?-
• ?- mitochdondrial
• ?- elongation
• ?- repair

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Function is Deduced From Activity
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Termination Least Well Understood

• Crowding would have to occur
• TBP- ter (termination site) binding protein -
  ?directs traffic?
• Yeast 5 (TxGy)n
• 3 (AxCy)n 100 bp
• DNA ligase seals nick between fragments by
  mechanism shown on next slides

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DNA Ligase is the Only Enzyme of Replication that
Requires Energy
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DNA Ligase is the Only Enzyme of Replication that
Requires Energy
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DNA Ligase is the Only Enzyme of Replication that
Requires Energy

• Mechanism analogous to attack on alpha
  phosphorous by polymerase

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Review of Replication - Initiation

• Ori bound by helicase and SSB (other proteins at
  site)
• Replication bubble forms
• primase and DNA polymerase III join complex
• synthesis is 5 to 3
• Okazaki fragments used on lagging strand
• Newer concept loop lagging strand

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Termination Least Well Understood

• Crowding would have to occur
• TBP- ter (termination site) binding protein -
  ?directs traffic?
• Yeast 5 (TxGy)n
• 3 (AxCy)n 100 bp
• DNA ligase seals nick between fragments
• Topoisomerase functions by similar mechanism,
  except E attaches to DNA instead of AMP (ATP not
  required)

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Review of Replication - Elongation

• PPi product
• polymerase I removes RNA and fills gap (in
  eukaryotes, Rnase H cuts out the RNA)

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Review of Replication - Termination

• Ligase seals nick (ATP dependent)
• Topoisomerase supercoils DNA (cut, wrap, ligate)

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Okazaki Fragments are Used to Synthesize the
Lagging StrandFig. 25-13 and 25-14 Lehninger POB
3rd Ed.

• synthesis is 5 to 3

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Okazaki Fragments are Used to Synthesize the
Lagging StrandFig. 25-13 and 25-14 Lehninger POB
3rd Ed.

• Newer model loop lagging strand

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Proofreading

• 1 mistake every 105 - 106 bases during
  replication
• (3 ? 5 exonuclease activity)
• In DNA, 1 mistake every 108 - 109 bases
• ?other repair mechanisms must exist

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Four Types of Repair Mechanisms

• Mismatch repair
• Base Excision repair
• Nucleotide Excision
• Direct Repair

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Mismatch Repair and dam

• ?which one is incorrect? crapshoot with 5050
  odds
• Upstream, GATC sequence, A methylated
• Takes a while for methylation to occur

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Base Excision Operates Where a Single Damaged
Base Occurs

• Uracil deglycosylase most common
• AP site
• AP endonuclease (variable specificity)

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Nucleotide Excision Operates in More Heavily
Damaged AreasFig. 25-23 Lehninger POB 3rd Ed.

• Removes more than just damaged ?because
  surroundings also likely to be damaged?

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Direct Repair of Thymidine Dimers by
PhotolyaseFig. 25-24 Lehninger POB 3rd Ed.
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In Eukaryotes, the Major Differences are the
Numbers and Names of the Molecules

• Prokaryotes Eukaryotes
• 1 ori and ter multiple ori and ter
• Polymerases III and I Pols d and a
• Okazaki fragments 1000 nt 100-250 nt
• supercoiling histones

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Types of Mutations

• I. Transition (Pu to other Pu)
• a. Silent (no change in amino acids)
• b. Missense (one amino acid converted to
  another)
• c. Nonsense (early termination of protein)
• II. Transversion (Pu to Py or vice versa)
• a. Silent, Missense or Nonsense
• III. Frame shift
• a. Insertion
• b. Deletion

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Genetic Rearrangement

• Random genetic drift

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Sister Chromatid Exchange... Fig. 38-11 Harpers
ROB 24th Ed.
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Occurs Through a Holliday IntermediateFig.
Lehninger POB Ed.
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Immunoglobulins Arise Through RecombinationFig.
59-7 Harpers ROB 24th Ed.
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VDJC Sequence Heavy ChainFig. 5-4 Stites, Stobo,
and Wells Basic and Clinical Immunology 6th Ed.
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VDJC Sequence Light ChainFig. 5-1 Stites, Stobo,
and Wells Basic and Clinical Immunology 6th Ed.
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Transposons are Jumping Genes

• Transposon short for transposable element
• Gene flanking regions
• Flanking region consists of inverted repeats

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Transposon Structure and Function

• Target site is simple and is cut to generate
  sticky ends

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Transposon Structure and Function

• Transponson is inserted, gaps filled in and
  ligated

The insertion can lead to a gene being turned on
(regulatory elements which will be discussed in
transcription) or turned off (gibberish produced).