Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 432

Biochemistry

Spring 2009

Lecture Notes: 23 March

© R. Paselk 2006
 
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DNA Replication, cont.

Eukaryotic DNA Synthesis

Mitotic Cell Cycle

In eukaryotes DNA replication in dividing cells takes place during a specific phase of the cell cycle, as noted below.

  • Mitosis (nuclear division with conservation of chromosome number) and cytokinesis (cell division) occur during the brief Mitotic (M) phase of the cell cycle. Mitosis is divided into five subphases in which the chromosomes are arranged and separated:
    1. Prophase
    2. Prometaphase
    3. Metaphase
    4. Anaphase
    5. Telophase and Cytokinesis
  • Mitosis is followed by the much longer Interphase in which cell growth and chromosome replication occur. Interphase is also divided into subphases. The biosynthesis of proteins, cell organelles etc., with the exception of DNA, occurs during all of the subphases.
    1. G1 (fist gap) phase. This is generally the longest phase of the cell cycle. Early in G1 proteins bind to the ORC (origin replication complex) to make a pre-replication complex (pre-RC). A variety of other proteins required for replication are also synthesized and/or form complexes during this phase to allow later DNA replication in the next phase.
    2. S phase is the only phase where DNA is synthesized and chromosomes duplicated.
    3. G2 (second gap) phase follows S phase. It is a relatively short phase in which additional growth takes place in preparation for the next cycle of mitosis.

       Eukaryotic Polymerases and DNA Synthesis

      Our studies will focus on the occurrences in S phase, beginning with DNA replication. As with prokaryotes, eukaryotes have a number of different polymerases specialized for different aspects of DNA replication and repair, as summarized in the table below:

      Polymerase Organelle Other Activities Structure Processivity Fidelity Inhibition
      alpha nucleus Primase 250 kD: tetramer of 180 kD core; 70 kD ?; 60 kD & 50 kD primase subs. Low, about 200 bp High Aphidicolin & N-Ethylmaleimide
      beta nucleus 36-38 kD Low Low Dideoxy NTP's
      gamma mitochondrian 3'-5' exonuclease 160-300 kD: 125 kD core; 35 kD; 47 kD High High Dideoxy NTP's & N-Ethylmaleimide
      del nucleus 3'-5' exonuclease 170 kD: dimer with 125 kD core, 50 kD sub which associates with PCNA. High High Aphidicolin & N-Ethylmaleimide, weak by Dideoxy NTP's
      epsilon nucleus 3'-5' exonuclease 256 kD: 215 kD core; 55 kD High High Aphidicolin & N-Ethylmaleimide, weak by Dideoxy NTP's

      The specific functions of these polymerases were established using specific inhibitors, combinations of which could block various polymerases while allowing others to continue.

      Polymerase alpha is a nuclear polymerase which participates in the replication of the chromosome, functioning in the initiation of DNA replication on the lagging strand. When provided with a ssDNA template it first synthesizes an RNA primer of about 10 nucleotides, then adds up to 20 or so deoxynucleotides. Note the very low processivity makes proofreading unnecessary.

      • In lagging strand synthesis, polymerasealpha first adds a primer and a stretch of about 20 deoxynucleotides. Synthesis is then switched (see below) to polymerase del (or epsilon), and additional deoxynucleotides are added. Priming happens frequently, occurring about every 50 nucleotides. The primer is removed, except for the last nucleotide, by RNase HI removes all but the last nucleotide, which is then hydrolyzed off by a complex called FEN1/RTH1. The gap is filled by polymerase, then the resulting Okazaki fragment is joined to the growing strand by DNA ligase.

       


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