DNA Replication

I. Possible modes of DNA replication
     A. Conservative
     B. Semiconservative
     C. Dispersive
II. Meselson and Stahl 1958
     A. CsCl density gradient
III. Harlequin chromosomes
     A. CHO cells
     B. 5-bromodeoxyuridine (BudR) -
          1. base analog
          2. substitutes for T
     C. Stain after 2 rounds of replication
          1. Giemsa and fluorescent dye
          2. One strand dark, other light
               a. two copies of BUdR is lighter
               b. one copy of BUdR is darker
               c. see picture p. 238
IV. Prokaryotic DNA replication
     A. Single origin of replication
          1. Replication bubble
          2. Both strands serve as templates
     B. Process
          1. Gyrase relaxes supercoils
          2. DNA wraps around initiator proteins
          3. DNA helicase binds and unwinds helix
          4. Single stranded binding protein (SSB)
               a. Binds to SS DNA during replication
               b. Keeps DNA from reannealing
     C. DNA primase
          1. Binds to helicase, forming complex called primosome
          2. All DNA polymerases can add nucleotides to preexisting strand
          3. DNA polymerases can't start if no nucleotide available to attach to
          4. Primase forms RNA primer - about 11 nucleotides long
     D. DNA polymerase  Arthur Kornberg 1955
          1. DNA polymerase I - repair
          2. DNA polymerase II - purpose unknown
          3. DNA polymerase III - replication
          4. New strand synthesized 5' to 3' by ALL polymerases
          5. Exonuclease function - proofreading, repair
     E. Replication fork
          1. Usually expands in both directions from initial replication bubble
          2. Two helicase molecules move in opposite directions
     F. Semidiscontinuous replication
          1. Leading strand - continuous
          2. Lagging strand - discontinuous
               a. Okazaki fragments
               b. Polymerase III stops when encounters RNA primer
               c. Polymerase I takes over, adding bases and simultaneously removing RNA nucleotides
               d. DNA ligase joins sugar phosphate backbone
V. Eukaryotic DNA replication (differences from prokaryotic)
     A. Occurs in S phase of cell cycle
     B. Multiple origins of replication
          1. Sequence of origin of replication well defined in Saccharomyces cerevisiae
          2. Autonomously replicating sequences
          3. Other eukaryotic sequences more complex and less well defined
     C. Replicon or replication unit
          1. Region replicated from one origin of replication
          2. Extends on both sides of origin
          3. To point where encounters replicated DNA from another origin
          4. Fig. 11.10, p. 247
     D. DNA polymerase
          1. Alpha, delta: replication
          2. Beta, epsilon: repair, proofreading
          3. Gamma: mitochondria, proofreading
     E. Replication at telomeres
          1. RNA primer at 5' end of new DNA strand
          2. Gap at 5' end of new strand
          3. DNA polymerase can't initiate strand synthesis at end
          4. Elizabeth Blackburn and Carol W. Greider - telomerase
               a. protein and RNA
               b. RNA complementary to DNA at end of telomere
               c. RNA serves as template, DNA is extended 5' to 3' (3 bases)
               d. RNA rejoins in different alignment; DNA extended further
               e. Process repeats several times; several 6 base repeats
               f. RNA primer made using extended DNA template
               g. DNA polymerase III uses RNA primer, extends chain
               h. DNA ligase joins backbone
               i. p. 249, fig. 11.12
          5. Repeats at telomere
     F. Assembly into nucleosomes
          1. Production of new histones; assembly into nucleosomes
          2. Coordinated with DNA replication
          3. Region of about 200-300 bp around replication forks that are nucleosome free
          4. Old nucleosomes remain intact
          5. Random attachment of old and new nucleosomes to the two strands
VI. DNA recombination
     A. Several steps
          1. Recognition and alignment
          2. SS break on each DNA molecule; each broken strand ‘invades' other molecule
          3. Gaps sealed to form Holliday intermediate structure
          4. Rotation of two double helices can occur, leading to
               a. Branch migration - movement of ‘crossover' point down molecule
          5. Heteroduplexes - two strands do not contain completely complementary sequences
          6. Holliday structure occurs when helices rotate to form a cross shape
          7. Cleavage of Holliday structure by endonuclease
               a. can lead to parental strands with inserted DNA (patched duplexes)
               b. can also produce recombinant strands (spliced duplexes)

For questions, comments and additional information, contact  mfhicks@pstcc.edu
Last Updated: June 24 2001
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