Problem of linear templates by qpv40869


									          Problem of linear templates
     3’         Replication
   5’                     3’

     5’                                  Primer?

• Since a primer is required, how do you initiate
  replication at the 5’ terminus of a DNA chain?
• How do you prevent progressive loss of DNA from
  the ends after replication?
Solutions to the problem of linear templates

 • Convert linear to circular DNA
 • Attach a protein to 5’ end to serve as primer
 • Make the ends repetitive, e.g. telomeres,
   and add more DNA after replication
           Telomerase adds repeats back to
                replicated telomeres
                                        Telomerase adds more
                +                       copies of "a’" to 3’ end of
                           aa           strand with overhang
    aaa                                                                 aa

The segment complementary to the             aaa                        a’a’a’a’
3’ end of template is not replicated.
                                                     DNA synthesis
          a = CCCCAA,
          a’ = GGGGTT                                                 a’a’a’a’
          in humans
      Replicated telomeres are primers for
re st of
               Telom er e
chrom osom e
                             (G T )n
                               4 2
...                                                             3'
...                                                             5'
                             ( C A ) n-2 or 3
                                4 2
                            shorten the C+A r ich ("bottom ") str and after replication

...                                                              3'
...                                                             5'
      Telomerase adds 1 nt at a time, using an
              internal RNA template
...                                                         3'
...                                                         5'

              Telom er ase adds new copies of s hort repe ats to the 3' end of the G+T rich ("top")
              strand, us ing an inter nal RNA as the tem plate.

...                                                       G
...                                                    AACCCCAACCCC

                                               3'                               inte rnal
                                                    telome ras e           5'   RNA
               Add 1 nucleotide at a tim e

...                                                       GG
...                                                    AACCCCAACCCC

                                                    telome ras e           5'
          Finish synthe sis of one re peat,
          and s hift over to s ynthesize the
...                                                        GGGGTTG
...                                                            AACCCCAACCCC

                                                                 telome ras e   5'
       Telomeric repeats form a primer for
      synthesis of the complementary strand
         After addition of mor e repeats, some specialized str uctur e for ms at the 3' end of
         the G+T rich strand involving G-G base pairs , e.g. a hair pin or a "G quartet."


          Pres um ably this structur e ser ves as the prim er for synthe sis of the C+A r ich str and


         Pr oces sing (as yet unidentified) would yie ld the extende d telom ere , w ith a
         short overhap by the G+T rich strand.

      Control of replication in bacteria
• Bacteria re-initiate replication more frequently
  when grown in rich media.
  – Doubling time of a bacterial culture can range
    from 18 min (rich media) to 180 min (poor media).
• Time required for replication cycle is constant.
  – C period
     • time to replicate the chromosome; 40 min
  – D period
     • time between completion of DNA replication
       and cell division; 20 min
  – C + D = 1 hour
  Multiple replication forks allow shorter
               doubling time
• Doubling time for a culture can vary, but
  time for replication cycle is constant!
• Variation is accomplished by changing the
  number of replication forks per cell.
• If doubling time of culture is < 60 min, then a
  new cycle of replication must initiate before
  the previous cycle is completed.
• Initiate replication at same frequency as cell
  doubling, e.g. every 30 min.
   forks in

E.g. every 30 min:
  Cells divide
  Replication initiates
           Cell cycle in eukarytoes

                                            S phase
         Preparation                        DNA synthesis
         for replication
                               6-8 hrs

                     ~12 hrs
 G0       G1                                  G2
                                  3-4 hrs
 cells    2nDNA                             4nDNA
   Multiple replicons per chromosome

• Many replicons per chromosome, with many
• Replicons initiate at different times of S
• Replicons containing actively transcribed
  genes replicate early, those with non-
  expressed genes replicate late.
         Regulation at check-points
• Critical check-points in the cell cycle are
  – G1 to S
  – G2 to M
• Passage is regulated by environmental
  signals acting on protein kinases
  – e.g., if enough dNTPs, etc for synthesis are
    available, then a signal activates a multi-
    subunit, cyclin-dependent protein kinase.
• Mechanism:
  – Increased amount of cyclin
  – Correct state of phosphorylation of the kinase
     More about cell cycle regulation
• BMB 460: Cell growth and differentiation
• BMB 480: Tumor viruses and oncogenes
• BMB/VSC 497A: Mechanisms of cellular

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