Chapter 10

Biosynthesis of Nucleic Acids - Replication

DNA metabolism

  • structure of DNA is fluid

  • new copy of DNA synthesized with high fidelity before cell division

  • errors are constantly checked for and repaired

  • segments are rearranged within a chromosome or between two DNA molecules giving offspring to a novel DNA

  • set of enzyme catalyzed and tightly regulated processes

    • DNA is the substrate that encodes its own metabolism

Flow of genetic information in the cell

  • Central Dogma: mechanisms by which information is transferred in the cell

  • Retroviruses: RNA as genetic material, RNA directs its own and DNA synthesis

Replication of DNA

  • naturally occurring DNA is single or double stranded, linear or circular

  • conservative, semiconservative, and dispersive replication possibilities

    • Meselson-Stahl Experiment

      • cells grown on 15N isotope then switched to 14N isotope and allowed to divide once

      • confirmed semiconservative replication

  • Fundamental rules:

    • Semiconservative (Meselson-Stahl)

    • begins at origin of replication and is bidirectional

      • circular DNA of prokaryotes: one origin, two forks

      • linear DNA of eukaryotes: several origins, two forks at each

    • catalyzed by DNA polymerase

      • nucleophilic attack by the 3’-OH of the growing DNA chain at the 5’-a-phosphate of the incoming dNTP

      • choice of dNTP (AGCT) is governed by existing template strand

        • DNA synthesis is template directed

      • all DNA polymerases require a template and a primer

        • primer: segment with free 3’-OH (primer terminus)

        • processivity: rate of synthesis divided by rate of dissociation

    • proceeds in 5’-3’ direction, semidiscontinuous

  • Challenges in replication

    • circular double stranded DNA

      • achievement of continuous unwinding and separation of two strands

      • protection of unwound portions from attack by nucleases

      • synthesis of DNA template strand from a 5’-3’ and a 3’-5’ strand

      • efficient protection from errors in replication

    • synthesis of leading and lagging strands

      • 5’-3’ direction on both strands but replication fork moves in one direction

      • leading strand synthesized continuously

      • lagging strand synthesized semi-discontinuously

        • okazaki fragments

          • DNA ligase: joins Okazaki fragments via covalent bonds

Enzymatic synthesis and degradation of DNA

  • 1. synthesis: DNA polymerase

    • E. coli has 5 DNA polymerases

    • function has all four deoxyribonucleoside triphosphates (ATGC), Mg++, RNA primer, DNA pol I, II, III

      • DNA pol I: repair and patching of DNA

      • DNA pol III: polymerization of newly formed DNA strand

      • DNA pol II, IV, V: proofreading and repair

    • Topoisomerase: remove and introduce supercoils

    • Helicase: promotes unwinding at replication fork

    • single stranded binding protein: stabilizes single stranded regions

    • primase: synthesis of RNA primers

      • catalyzes copying of short stretch of DNA template to produce RNA primer sequence

    • DNA ligase: covalently link Okazaki fragments

    • synthesis and linking of new DNA strands is begun by DNA pol III

      • new DNA is linked to 3’-OH of RNA primer

      • replication fork moves away, RNA primer is removed by DNA pol I

  • 2. synthesis: reverse transcriptase

  • 3. degradation: nucleases

    • exonucleases: degrade DNA from one end, direction specific

    • endonucleases: degrade DNA from internal sites, non-specific or specific

DNA Replication

  • Initiation

    • begins at OriC (245 bp, highly conserved)

  • Elongation

    • occurs at replication fork with leading and lagging strands

  • Termination

    • Ter sites and Tus protein

      • bind together and block activity of helicase

        • traps one replication fork at Tus-Ter complex

    • two replication forks can collide

      • causes DNA damage

DNA replication in eukaryotes

  • chromosome is larger, more complex, and linear

  • regulation is more complex - tied to cell cycle

  • rate of replication is slower

  • multiple sites for origin of replication

Error rates of DNA replication

  • Errors = mutations

  • E. coli: errors are rarely made

  • template directed synthesis works very well

  • incorrect bases do not base pair properly with template and are rejected before bond is made

    • rare tautomers can hydrogen bond to wrong base

  • 3’-5’ exonuclease activity of DNA polymerase removes mismatched base and fills in the correct base

    • proofreading: removal of incorrect nucleotides immediately after they are added to the growing DNA

DNA repair

  • cells usually have one or two copies of genomic DNA

    • genetic information inherent in nucleotide sequence must be maintained

  • RNA and proteins turnover in cell quickly

    • mistakes in RNA and proteins are often minor

  • Genomic DNA does not turnover, it is replicated

    • mistakes in DNA are permanent = mutations

    • mutation: permanent change in nucleotide sequence of the genome

  • types of mutations

    • substitution mutation: replacement of one base with another

    • insertion or deletion mutation: addition or loss of one or more bases

    • silent mutation: mutation that has no effect on gene function

  • DNA lesions produced by chemistry of nucleotides and environmental damage

Non-enzymatic reactions of nucleobases

  • deamination or loss of exocyclic amino groups

  • hydrolysis of glycosidic bond

    • more common for purines

    • loss of purine = apurinic site

    • loss of pyrimidine = apyrimidinic site

  • depurination

  • light induced DNA damage