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14 DNA REPLICATION (Full Edition)

1 Introduction

Key Terms
  • The replisome is the multiprotein structure that assembles at the bacterial replication fork to undertake synthesis of DNA. It contains DNA polymerase and other enzymes.
  • A dna mutant of bacteria is temperature-sensitive; it cannot synthesize DNA at 42°C, but can do so at 37°C.
  • A quick-stop mutant is a type of DNA replication temperature-sensitive mutant (dna )in E. coli that immediately stops DNA replication when the temperature is increased to 42°C.
  • A slow-stop mutant is a type of DNA replication temperature-sensitive mutant in E. coli that can finish a round of replication at the unpermissive temperature, but cannot start another.
  • In vitro complementation is a functional assay used to identify components of a process. The reaction is reconstructed using extracts from a mutant cell. Fractions from wild-type cells are then tested for restoration of activity.

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Replication of duplex DNA is a complex endeavor involving a conglomerate of enzyme activities. Different activities are involved in the stages of initiation, elongation, and termination.

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  • Initiation involves recognition of an origin by a complex of proteins. Before DNA synthesis begins, the parental strands must be separated and (transiently) stabilized in the single-stranded state. Then synthesis of daughter strands can be initiated at the replication fork.
  • Elongation is undertaken by another complex of proteins. The replisome exists only as a protein complex associated with the particular structure that DNA takes at the replication fork. It does not exist as an independent unit (for example, analogous to the ribosome). As the replisome moves along DNA, the parental strands unwind and daughter strands are synthesized.
  • At the end of the replicon, joining and/or termination reactions are necessary. Following termination, the duplicate chromosomes must be separated from one another, which requires manipulation of higher-order DNA structure.

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Inability to replicate DNA is fatal for a growing cell. Mutants in replication must therefore be obtained as conditional lethals. These are able to accomplish replication under permissive conditions (provided by the normal temperature of incubation), but they are defective under nonpermissive conditions (provided by the higher temperature of 42°C). A comprehensive series of such temperature-sensitive mutants in E. coli identifies a set of loci called the dna genes. The dna mutants distinguish two stages of replication by their behavior when the temperature is raised (545):

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  • The major class of quick-stop mutants cease replication immediately on a temperature rise. They are defective in the components of the replication apparatus, typically in the enzymes needed for elongation (but also include defects in the supply of essential precursors).
  • The smaller class of slow-stop mutants complete the current round of replication, but cannot start another. They are defective in the events involved in initiating a cycle of replication at the origin.

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An important assay used to identify the components of the replication apparatus is called in vitro complementation. An in vitro system for replication is prepared from a dna mutant and operated under conditions in which the mutant gene product is inactive. Extracts from wild-type cells are tested for their ability to restore activity. The protein coded by the dna locus can be purified by identifying the active component in the extract.

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Each component of the bacterial replication apparatus is now available for study in vitro as a biochemically pure product, and is implicated in vivo by mutations in its gene. Eukaryotic replication systems are highly purified, and usually have components analogous to the bacterial proteins, but have not necessarily reached the stage of identification of every single component.

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reviews
  • 545 Hirota, Y., Ryter, A., and Jacob, F. (1968).  Thermosensitive mutants of E. coli affected In the processes of DNA synthesis and cellular division.  Cold Spring Harbor Symp. Quant. Biol. 33, 677-693.  PubMed  

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