Rfc Form Template

							DNA REPLICATION:
 Eukaryotes and
     Phages
      Paul D. Brown, PhD
 paul.brown@uwimona.edu.jm
  BC21C: Molecular Biology I
Replication in eukaryotes
Mammalian DNA polymerases
Eukaryotic Replication Machinery

 • Eukaryotic machinery is generally similar to that of E.
   coli.
 • Can get virus SV40 to replicate in vivo with 8 different
   purified components from mammalian cells. (SV40
   virus, causes cancer, is model for eukaryotic
   replication)
 • Replication occurs bidirectionally with RNA primers. It
   is semidiscontinuous.
 • But two distinct DNA polymerases work at the growing
   fork, a and either d or e.
The eukaryotic replication machinery is
   generally similar to that of E. coli




                                 Figure 12-12
1. The SV40 encoded protein T-antigen (T-ag) binds to the
   SV40 origin, melting it with its helicase activity; requires
   ATP. Local melting occurs then the hexameric form of T-
   ag binds, which has helicase activity.
2. It binds around both leading strands (NB there are two).
3. Replication protein A (RPA), a host cell SSB binds to
   the ssDNA. One molecule of DNA pol a, tightly bound to
   primase, binds to each template strand. Primases form
   the RNA templates, these are elongated for a short
   stretch by Pol a. These form the origins of the two
   leading strands. The activity of Pol a is stimulated by
   replication factor C (RFC).
4. Binding of PCNA (proliferating cell nuclear antigen) at
   the primer template 3’ termini displaces Pol a, this
   interrupts leading strand synthesis.
5. Pol d associates with PCNA, which increases the
   processivity of the enzyme and it makes the rest of the
   leading strand. PCNA is analogous to the b clamp of E.
   coli pol III. PCNA is a trimer, not a dimer. As T-ag
   helicase progresses along from the origin, primase-pol a
   complex associates with the melted template,
   downstream from the leading strand primers. Synthesis
   of the lagging strand is carried out by combination of
   primase, Pol a and RFC.
Eukaryotic DNA polymerases: PRIMASE
   DNA Pol a(I):primase complex- bifunctional

                         Heterotetrameric phosphoprotein

      48 kDa PRIMASE- initiates DNA synthesis (makes
      complementary RNA primer)

      58 kDa protein- tethers primase to 180 kDa subunit

      180 kDa polymerase A subunit- extends RNA primer
      by making a short DNAi (3-4 bases) (only in Drosophila
      has proofreading activity

      70 kDa subunit- no known catalytic function
      (may recruit pola:primase to the replication fork)
    Eukaryotic DNA Replication:
1. DNA pol a (I) + 2 primase subunits: initiate
  synthesis of lagging and leading strands. RNA-
  DNAi (3-4 b) primer.
2. DNA pol d (III) : elongates both lagging and
  leading strands.
3. PCNA: (proliferating cell nuclear antigen) acts as
  processivity factor for leading strand (like b
  clamp); elongation.
4. Replication factors C (RF-C; clamp loading,
  ATPase) and RF-A (single strand binding).
5. Topoisomerases I & II: maintains DNA winding.
Similar functions at bacterial and mammalian
replication forks:
 Function            E. coli        HeLa/SV40

 •helicase           DnaB            T antigen
 •loading helicase   DnaC            T antigen
 • single strand     SSB             RF-A
 • priming           DnaG            Pol a(I)/2primases
 • sliding clamp     b               PCNA
 • clamp loading     gd              RF-C
 • catalysis         Pol III core    Pol d(III)
 • holoenzyme        t               ???
 dimerization
 • RNA removal       Pol I           MF 1 exonuclease
 • ligation          ligase          ligase 1
           Minimal oriC
                            DnaA binding

Region of melting

    L M     R       1                  2     3     4



        13-mers                    9-mers
                          245 bp

  GATCTNTTNTTTT                    TTATNCANA

                        Note: GATC is Dam methylation site
          E. coli has a covalently
    closed circular chromosome
4 DNA polymerase
complexes (2 per
replication fork) at
oriC


Proceed in opposite
directions and meet
at opposite side.


Circular daughter
chromosomes are
untangled
       Eukaryotic chromosomes
              are linear
DNA primase can not
start at an end
Ends are called
telomeres
Repetitive sequence
GGGGTTA

Telomerase required
to complete the ends


DNA-RNA dependent
DNA polymerase
          Rolling circle replication
• Used by bacteriophage and Xenopus laevis.
• This is applicable to circular DNA
• Nick opens in ds DNA, 3’ end is extended, displacing the
  original parent strand.
Rolling circle replication is
commonly used by
bacteriophages. Monomers
of the genome are cleaved
off and packaged. Phage
fX174 has a ssDNA genome
known as plus strand. When
it replicates, the – strand is
made to make the ds duplex.
This replicated by the rolling
circle mechanism.
• When the duplex is made, it is covalently closed and it
  becomes supercoiled. The phage coded A protein nicks
  the + strand at the origin. The A protein remains
  attached to the 5’ end while the 3’ end is extended. The
  DNA can only be nicked when it is supercoiled.
• The A protein binds to a ssDNA decamer at the site of
  this nick. An enzyme (relaxase) binds to supercoiled
  DNA, cleaves it and binds to the released 5’ end. The 3’
  OH end of the nick is extended into a new chain. The
  chain is elongated around until it reaches the start point
  again.
• The A protein stays with the rolling circle as well as to
  the 5’ end of the displaced tail as it passes, so it is able
  to recognise the origin again as it passes. When it does,
  the A protein nicks it, attaching to the new 5’ end. The A
  protein is involved in the circularisation process.
    174 phage as a simple model for replication:


                                Rolling
             “Lagging           circle
             strand”            replication
             synthesis                        (+)
 (+)

       + strand   Replicative
                                                    (+)
                  form (RF):
                  ds plasmid

                                               + strand packaged
                                               to form virion
Rolling circle replication is a model
for leading strand synthesis.
174 phage replication provides a model for DNA
replication
      RF
 (randomly nicked)           (+)          SSB =        = Rep (helicase)

               (+)
       (-)
                                                   +
                                           (+)                 (-)
    Background for
    Rolling circle
    replication:     Note: no DNA polymerase just to separate single strands

      Two kinds of activities are needed to convert double-
      stranded RF DNA to single-stranded DNA without
      synthesis of new DNA.
      a. Helicase: separates the strands using ATP to provide the
      energy.
      b. single-strand binding protein. (SSB).
       174 as a simple model for replication
                                                 DNA pol III elongates
  Rolling circle                                 3’-end of the nick.
  replication:                 SSB
            Gene A      (+)              (+)
Rep                                                                      (+)
            protein                                               +
(+)
      (-)                (-)               (-)              (-)


      Proteins needed for rolling circle replication:
      a. “gene A” protein to nick at origin (pas). Covalently linked to 5’end
      of the displaced strand.
      b. SSB protein to keep DNA single-stranded. Binding is highly
      cooperative.
      c. Rep protein provides helicase function.
      d. DNA pol III holoenzyme
How would a dsDNA
 phage replicate?