Replication RNA Synthesis Decoding the Genetic Code Noel Murphy Reference Sources Hartl Jones Genetics Analysis of Genes and Genomes 6th Edi

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Replication RNA Synthesis Decoding the Genetic Code Noel Murphy Reference Sources Hartl Jones Genetics Analysis of Genes and Genomes 6th Edi Powered By Docstoc
					       Replication
     RNA Synthesis
Decoding the Genetic Code

                     Noel Murphy
            Reference Sources

Hartl & Jones, Genetics: Analysis of Genes and Genomes,
6th Edition
Chapter 6 – Replication
Chapter 10 – Transcription and the code
Klug & Cummings, Essentials of Genetics 5th Edition
Chapter 11 – Replication
Chapter 13 – Transcription and the code
Lectures http://www.tcd.ie/Genetics/staff/Noel_Murphy.htm
    DNA is the Genetic Material
Therefore it must
(1) Replicate faithfully.
(2) Have the coding capacity to generate
    proteins and other products for all
    cellular functions.

• “A genetic material must carry out two jobs:
  duplicate itself and control the development of the
  rest of the cell in a specific way.”
                                        • -Francis Crick
Replication
The Dawn of Molecular Biology
              April 25, 1953
Watson and Crick: "It has not escaped our
notice that the specific (base) pairing we
have postulated immediately suggests a
possible copying mechanism for the
genetic material."
        Models for DNA replication
1) Semiconservative model:
Daughter DNA molecules contain one parental
strand and one newly-replicated strand

2) Conservative model:
Parent strands transfer information to an
intermediate (?), then the intermediate gets copied.
The parent helix is conserved, the daughter
helix is completely new

3) Dispersive model:
Parent helix is broken into fragments, dispersed,
copied then assembled into two new helices.
New and old DNA are completely dispersed
     MODELS OF DNA REPLICATION
(a) Hypothesis 1:    (b) Hypothesis 2:         (c) Hypothesis 3:
Semi-conservative   Conservative replication   Dispersive replication
   replication




                     Intermediate molecule
Testing Models for DNA replication
  Matthew Meselson and Franklin Stahl (1958)
   Meselson and Stahl
     Semi-conservative replication of DNA




Isotopes of nitrogen (non-radioactive) were used in this experiment
Generations
    0           HH
                                                      Equilibrium Density
                                                      Gradient
    0.3                                               Centrifugation
                                            HH
                                                      Detection of
                                                      semiconservative
    0.7                                               replication in E. coli
                                                      by density-gradient
                                                      centrifugation. The
    1.0         HL                                    position of a band of
                                                      DNA depends on its
    1.1                                               content of 14N amd
                                                      15N. After 1.0

                                                      generation, all the
                                  HL                  DNA molecules are
    1.5                                               hybrids containing
                                                      equal amounts of 14N
                                                      and 15N
    1.9       LL + HL


    2.5




    3.0


    4.1


 0 and 1.0
  mixed


 0 and 4.1
  mixed                 HL   LL        LL        LH
                        DNA replication
Nucleotides are successively added using deoxynucleoside triphosphosphates (dNTP’s)
          Replication as a process
• Double-stranded DNA unwinds.

The junction of the unwound
molecules is a replication fork.

A new strand is formed by pairing
complementary bases with the
old strand.

Two molecules are made.
Each has one new and one old
DNA strand.
               DNA Replication
• Since DNA replication is semiconservative, therefore
  the helix must be unwound.

• John Cairns (1963) showed that initial unwinding is
  localized to a region of the bacterial circular genome,
  called an “origin” or “ori” for short.
        Replication can be Uni- or Bidirectional



     UNIDIRECTIONAL REPLICATION
           Origin
                               BIDIRECTIONAL REPLICATION
5’                                3’     Origin
3’                                5’

                          5’                               3’
                          3’                               5’
                                                John Cairns
    Bacterial
    culture
     *T        *T

          *T        *T


                                    Grow cells for several generations                 *T
in media with low                    Small amounts of 3H thymidine
                                                                                            *T    *T



concentration of                     are incorporated into new DNA
  3H- thymidine                                                                    All DNA is lightly
                                                                               labeled with radioactivity

                                                                                                   Add a high
                                                                                                  concentration
                                                                     Grow for brief              of 3H- thymidine
                                                                     period of time
                                            *T *T  *T      *T*T
                                    *T *T       *T    *T       *T
                                              *T *T       *T
                                           *T                   *T
                                                 *T *T *T
                                     *T
                                        *T *T *T *T *T *T *T *T




                         Dense label at the replication fork
                          where new DNA is being made


Cairns then isolated the chromosomes by lysing the cells very very gently
and placed them on an electron micrograph (EM) grid which he exposed to
X-ray film for two months.
Evidence points to bidirectional replication




                Label at both replication forks
   Features of DNA Replication
• DNA replication is semiconservative
  – Each strand of both replication forks is being
    copied.


• DNA replication is bidirectional
  – Bidirectional replication involves two
    replication forks, which move in opposite
    directions
         Arthur Kornberg (1957)
                       Protein extracts from E. coli
                                    +
                              Template DNA
                       Is new DNA synthesized??


                     - dNTPs (substrates) all 4 at once
                     - Mg2+ (cofactor)
                     - ATP (energy source)
                     - free 3’OH end (primer)
                     In vitro assay for DNA synthesis


Used the assay to purify a DNA polymerizing enzyme
                 DNA polymerase I
 Kornberg also used the in vitro assay to characterize
 the DNA polymerizing activity

 - dNTPs are ONLY added to the 3’ end of newly
 replicating DNA

5’              3’ New progeny strand
3’                                      5’ Parental template strand

5’                3’
3’                                      5’


5’                     3’
3’                                      5’

5’                          3’
3’                                      5’




 -therefore DNA synthesis occurs only in the
              5’ to 3’ direction
THIS LEADS TO A CONCEPTUAL PROBLEM

 Consider one replication fork:

                                         3’
                                            5’
                                         Primer
                                    3’


                          Continuous replication
5’
 Direction of
 unwinding
3’              5’
                          Discontinuous replication
                     3’
                          5’
                               3’


                                         5’
Evidence for the Semi-Discontinuous replication
  model was provided by the Okazakis (1968)
  Evidence for Semi-Discontinuous Replication
           (pulse-chase experiment)


               Add 3H Thymidine Flood with non-radioactive T
 Bacterial                                             Harvest the bacteria
 culture                                                 at different times
               For a SHORT time    Allow replication
               (i.e. seconds)        To continue          after the chase

Bacteria are
replicating

                                           smallest

 Isolate their DNA                                     Radioactivity will only
 Separate the strands                                 be in the DNA that was
 (using alkali conditions)                            made during the pulse
 Run on a sizing gradient
                                            largest
         Results of pulse-chase experiment

                               Pulse
                     Chase
                                            3’
                                               5’
                                            Primer
                                       3’


                                                     smallest

5’
 Direction of
 unwinding
3’              5’
                     3’
                                                     largest
                          5’
                                 3’
                                  ***
                                            5’
DNA replication is semi-discontinuous


  Continuous synthesis




  Discontinuous synthesis
     Features of DNA Replication
• DNA replication is semiconservative
   – Each strand of template DNA is being copied.
• DNA replication is bidirectional
   – Bidirectional replication involves two replication forks,
     which move in opposite directions
• DNA replication is semidiscontinuous
   – The leading strand copies continuously
   – The lagging strand copies in segments (Okazaki fragments)
     which must be joined
             The Enzymology
                of DNA Replication

• In 1957, Arthur Kornberg demonstrated the
  existence of a DNA polymerase - DNA
  polymerase I
• DNA Polymerase I has THREE different
  enzymatic activities in a single polypeptide:
• a 5’ to 3’ DNA polymerizing activity
• a 3’ to 5’ exonuclease activity
• a 5’ to 3’ exonuclease activity
       The 5’ to 3’ DNA polymerizing activity




Subsequent
hydrolysis of
PPi drives the
reaction forward




            Nucleotides are added at the 3'-end of the strand
        Why the exonuclease activities?

• The 3'-5' exonuclease activity serves a
  proofreading function
• It removes incorrectly matched bases, so that
  the polymerase can try again.
Proof reading activity
of the 3’ to 5’ exonuclease.

DNAPI stalls if the incorrect
ntd is added - it can’t add the
next ntd in the chain


Proof reading activity is slow
compared to polymerizing
activity, but the stalling of
DNAP I after insertion of an
incorrect base allows the
proofreading activity to
catch up with the polymerizing
activity and remove the
incorrect base.
     DNA Replication is Accurate
    (In E. coli: 1 error/109 -1010 dNTPs added)

How?
1) Base-pairing specificity at the active site
- correct geometry in the active site occurs only with
correctly paired bases BUT the wrong base still gets
inserted 1/ 104 -105 dNTPs added
2) Proofreading activity by 3’-5’ exonuclease
- removes mispaired dNTPs from 3’ end of DNA
- increases the accuracy of replication 102 -103 fold
3) Mismatch repair system
- corrects mismatches AFTER DNA replication
Is DNA Polymerase I the principal
     replication enzyme??
 In 1969 John Cairns and Paula deLucia isolated a
     mutant bacterial strain with only 1% DNAP I
                     activity (polA)
- mutant was super sensitive to UV radiation
- but otherwise the mutant was fine i.e. it could
   divide, so obviously it can replicate its DNA
Conclusion:
• DNAP I is NOT the principal replication
  enzyme in E. coli
               Other clues….
- DNAP I is too slow (600 dNTPs added/minute –
  would take 100 hrs to replicate genome instead of
  40 minutes)
- DNAP I is only moderately processive
  (processivity refers to the number of dNTPs added
  to a growing DNA chain before the enzyme
  dissociates from the template)

Conclusion:
• There must be additional DNA polymerases.
• Biochemists purified them from the polA mutant
 So if it’s not the chief replication enzyme
         then what does DNAP I do?


- functions in multiple processes that require
   only short lengths of DNA synthesis
- has a major role in DNA repair (Cairns-
   deLucia mutant was UV-sensitive)
- its role in DNA replication is to remove
   primers and fill in the gaps left behind
- for this it needs the nick-translation activity
     The DNA Polymerase Family
A total of 5 different DNAPs have been reported
                       in E. coli
• DNAP I: functions in repair and replication
• DNAP II: functions in DNA repair (proven in
  1999)
• DNAP III: principal DNA replication enzyme
• DNAP IV: functions in DNA repair (discovered in
  1999)
• DNAP V: functions in DNA repair (discovered in
  1999)
            DNA Polymerase III
      The "real" replicative polymerase in E. coli
• It’s fast: up to 1,000 dNTPs added/sec/enzyme
• It’s highly processive: >500,000 dNTPs added
  before dissociating
• It’s accurate: makes 1 error in 107 dNTPs added,
  with proofreading, this gives a final error rate of 1
  in 1010 overall.

				
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