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SEPARATING THE TWO TEMPLATE DNA STRANDS

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To serve as templates for DNA replication, the two DNA strands must be separated, a task that DNA polymerase cannot perform on its own. The strands must be separated because the bases of the DNA are inside the double helix, where they are not available to pair with the incoming deoxynucleotides to direct which nucleotide will be inserted at each step. Proteins called DNA helicases separate the strands of DNA (see Singleton et al., Suggested Reading). Many of these proteins form a ring around one strand of DNA and propel the strand through the ring, acting as a mechanical wedge that strips the strands apart as it moves. It takes a lot of energy to separate the strands of DNA, and helicases cleave a lot of ATP for energy, forming ADP in the process. There are about 20 different helicases in E. coli, and each helicase works in only one direction, either the 3′-to-5′ or the 5′-to-3′ direction. The DnaB helicase that normally separates the strands of DNA ahead of the replication fork in E. coli is a large doughnut-shaped complex composed of six polypeptide products of the dnaB gene. It propels one strand, the template for lagging-strand DNA replication, through the center of the complex in the 5′-to-3′ direction, opening strands of DNA ahead of the replication fork (Figure 1.10). The DnaB ring cannot load onto single-stranded DNA on its own to start a DNA replication fork; it requires the loading protein DnaC. Other helicases are discussed in later chapters in connection with recombination and repair.

Once the strands of DNA have been separated, they also must be prevented from coming back together (or from annealing to themselves if they happen to be complementary over short regions). Separation of the strands is maintained by proteins called single-strand-binding (SSB) proteins or, less frequently, helix-destabilizing proteins. They are proteins that bind preferentially to singlestranded DNA and prevent double-stranded helical DNA from reforming prematurely. Interestingly, SSB activity goes beyond this passive role. SSB is also responsible for recruiting a number of replication and repair proteins through a specific set of amino acids encoded in the very C-terminal end of SSB, allowing it to serve as an organizational hub for other processes.

Snyder and Champness Molecular Genetics of Bacteria

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