Bio3400 Chapter 11 DNA Replication and Recombination
  1. The                  of DNA strands allows each strand to serve as a           for synthesis of the other.

      DNA is synthesized by unwinding the helix, then using base-pairing rules to replicate each strand. The process is semiconservative: each replicated double helix consists of one "old" and one "new" strand.


      The 2 DNA strands run in opposite (antiparallel) directions; one in the 3' - 5' direction, the other 5' - 3'. The A-T and G-C base pairing provides complementarity of the two strands. Thus, DNA follows these base-pairing rules: A always pairs with T, and G always pairs with C.
     
     
     
     
  2. DNA replication in bacteria begins at the         of replication and is                .

      Bidirectional replication of the E. coli chromosome starts at a fixed origin of replication (oriC). As the DNA unwinds, two replication forks (arrows) move away from the origin, forming a replication bubble. The forks merge as DNA replication is completed at a termination region (ter), reproducing one replicon.
     
     
     
     
    • The DNA helix is unwound by proteins called            which bind to the origin of replication and break the           bonds between the bases.

        Unwinding of the bacterial helix begins when monomers of the helicase protein DnaA bind to DNA sites containing repeating sequences of 9 and 13 bases (called 9mers and 13mers). continue


        DnaB and DnaC helicase proteins open the helix by breaking hydrogen bonds between the bases, denaturing the double helix and forming a replication bubble. Energy to break the hydrogen bonds is provided by the hydrolysis of ATP.
       
       
       
       
    • Producing of DNA polymers requires a      primer made by the enzyme          .

        Initiation of DNA synthesis begins when primase builds a short RNA primer in the 5' to 3' direction that is complementary to the template strand of the helix.
       
       
       
       
    • Polymerization is catalyzed by several DNA polymerases.

        These DNA polymerases cannot initiate DNA synthesis, but can elongate an existing DNA or RNA strand (primer). All three possess 3' to 5' exonuclease activity which allows proofreading to correct mismatched base pairs. DNA polymerase I fills gaps in the DNA and also removes the primer by its 5' to 3' exonuclease activity. Polymerases II is involved in DNA repair. Polymerase III is the main enzyme for 5' to 3' polymerization in vivo.


        The active form of DNA polymerase III is called a holoenzyme, a dimer with 10 different polypeptide subunits. The a (alpha), e (epsilon) and th (theta) subunits make up the core enzyme to perform polymerization and proofreading. The g (gamma) complex is involved in "loading" the enzyme onto the template at the replication fork. The b (beta) subunit serves as a "clamp" and prevents the core enzyme from falling off the template during polymerization. The t (tau) subunit functions to dimerize two core polymerases allowing simultaneous synthesis of both strands of the helix. The holoenzyme and several other proteins at the replication fork together form a huge complex called the replisome.
       
       
       
       
    • Chain             occurs in the 5' to 3' direction by addition of one nucleotide at a time to the 3' end.

        DNA elongation. The precursor dNTP (nucleoside triphosphate) contains 3 phosphate groups attached to the 5'-carbon of deoxyribose. As the 2 terminal phosphates are cleaved, the remaining phosphate is linked to the 3'-OH group of the chain. Thus, chain elongation occurs in the 5' to 3' direction by adding a nucleotide to the 3' (-OH) end.
       
       
       
       
    • DNA synthesis is continuous along the          strand of the replication fork, but is discontinuous along the          strand. Synthesis is             on both strands.

        Since polymerization by DNA polymerase III occurs only in the 5' to 3' direction, elongation along the two antiparallel strands are dissimilar. Synthesis along the leading strand of a replication fork can occur continuously. Synthesis along the lagging strand must be discontinuous, occurring in Okazaki fragments, each with an RNA primer.


        Polymerization occurs concurrently on both strands by a single DNA polymerase III holoenzyme. The lagging template strand is looped at the replication fork, allowing each core enzyme of the dimer to add bases in the 5' to 3' direction.


        The b (beta) subunit forms a dimer that serves as a "clamp" to keep the core enzyme bound to the DNA templates. Thus the entire holoenzyme moves along the parent duplex as a sliding clamp, advancing the replication fork.
       
       
       
       
    • The RNA primer is removed by                   which replaces it with DNA bases.

        DNA polymerase I fills gaps in the synthesized strand and also removes the primer by its 5' to 3' exonuclease activity.
       
       
       
       
    • The Okazaki fragments are joined together by DNA         , which forms the missing phosphodiester bonds between the fragments.
     
     
     
     
    Prokaryote DNA synthesis summary.

      Summary of DNA synthesis in bacteria.
    • At the advancing fork, helicase proteins unwind the double helix.
    • Primase lays down a short RNA primer.
    • Each core enzyme of the DNA polymerase III dimer is bound to a template strand by a beta-subunit sliding clamp.
    • Synthesis is continuous on the leading strand, but occurs in Okazaki fragments on the lagging strand.
    • The lagging strand loops around to allow concurrent synthesis on both strands.
    • DNA polymerase I replaces the RNA primer (synthesized by primase) with DNA.
    • DNA ligase joins the Okazaki fragments.
     
     
     
     
  3. Eukaryotic DNA synthesis is more complex than that in prokaryotes.
       
       
       
       
    • Eukaryotic genomes are         and contain multiple origins of replication to allow the genome to be replicated in a few hours.


          Eukaryotic chromosomes contain multiple replication origins that form multiple replication bubbles that are replicated in multiple replicons. The origins reside within an AT-rich regions, where a helicase enzyme unwinds the double helix.
         
         
         
         
      • Several DNA              have been discovered; four partake in replication, the rest are involved in         processes.


        Three eukaryotic DNA polymerases catalyze reactions in DNA replication, while others are involved in repair. Pol a (alpha) synthesizes the RNA primers during initiation. Then, in a process called polymerase switching, it is replaced by Pol d (delta), which performs the main task of concurrent elongation of both strands. Pol e (epsilon) is the other enzyme involved in nuclear DNA synthesis, possibly playing a role in binding to the origin or synthesis of the lagging strand. Pol g (gamma) is encoded by a nuclear gene though its function is synthesis of mitochondrial DNA.
       
       
       
       
    • Eukaryotic chromosomes are complexed with           , forming              that also need to be duplicated during DNA synthesis.

        Eukaryotic chromosomes are associated with proteins called histones, forming complexes of nucleosomes. The histones also need to be duplicated, and then reassociated with DNA into nucleosomes during the S phase of the cell cycle.


        In a typical somatic cell cycle, the period of active cell division is mitosis and the interval between cell divisions is interphase. After mitosis, the cells enters the first gap phase, G1, and may then become nondividing (G0) or continue to S (DNA synthesis), G2, and undergo mitosis again.
       
       
       
       
    • Eukaryotic chromosomes are         rather than circular, and pose a problem when terminating replication on the          strand.


          Semiconservative synthesis of the leading strand in a linear chromosome can proceeds normally to the end of the double helix. On the lagging strand, after the last RNA primer is removed, there is no free 3'-OH for DNA polymerase to elongate. The gap on the lagging strand leads to shortening of the chromosome after each round of synthesis. This chromosome shortening may play a role aging of somatic cells, and must be avoided in germ cells.
         
         
         
         
      • Repeating sequences of DNA called            are synthesized by the enzyme             to prevent chromosome shortening in       cells.


        The enzyme telomerase can synthesize short DNA sequences (telomeres) at the 3' end of eukaryotic chromosomes, preventing chromosome shortening in germ cells. The enzyme adds repeats of TTGGGG sequences that fold back on themselves by forming unorthodox G-G hydrogen bonds. The gap is filled by a DNA polymerase and ligase. The hairpin loop is then cleaved off, preserving the original duplex. This allows gametes and malignant cells, as well as some "immortal" cultured cells, to continue duplicating the linear DNA.


        Transmission electron micrograph of human DNA from a HeLa cell, illustrating replication forks and the associated replication bubble. HeLa cells were derived from cervical cancer cells taken from "Helen Lane", who died from her cancer in 1951, but her cells, which possess high telomerase activity, have continued to divide in culture.
       
       
       
       
    • Homologous                in general involves DNA exchange along two chromosomes with sequence homology.

        Homologous recombination during meiosis.
      • Two DNA duplexes that share homologous sequence are paired.
      • Endonuclease nicking produces single-stranded cuts at identical positions.
      • The single strands are displaced and pair with their complements on the opposite duplex.
      • A ligase seals the loose ends, creating heteroduplex DNA molecules. 2 3 4


        Homologous recombination.
      • Branch migration lengthens the heteroduplex as hydrogen bonds are broken and reformed along each duplex.
      • The duplexes now separate.
      • continue


        Homologous recombination.
      • The bottom portions rotate 180°, creating a planar x (chi) form (Holliday structure ).
      • The other two strands are now nicked by an endonuclease.
      • continue


        Homologous recombination.
      • The nicks are close by ligase, creating recombinant duplexes.
       
       
       
       
    • DNA recombination can also occur by gene             , caused by excision repair of a heteroduplex in meiosis.


      A base-pair mismatch occurs in one of the two homologs during heteroduplex formation in meiosis. During excision repair, one of the two mismatches is removed and the complement is synthesized, leading to possible gene conversion.