Lecture13S10

Lecture13S10 - BIS101/Engebrecht Lecture 13 4/29/10 The...

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BIS101/Engebrecht Lecture 13 4/29/10 The basic mechanism underlying DNA replication is the addition of a nucleotide (the polymerase uses dNTP) to the 3’-OH group of the sugar from a primer nucleotide in a template directed manner using Watson/Crick base-pairing principles. DNA grows in a 5’ 3’ direction. Not surprisingly, this process requires a large number of gene products, central of which is DNA polymerases. DNA-dependent DNA polymerases can only: 1) elongate a chain in one direction, 5’ 3’. 2) These enzymes need a 3’-OH group for phosphodiester bond formation. They cannot form a new strand on a template de novo, they must have a primer (a short stretch of nucleotides). 3) They need a template molecule for which to copy. We can consider four major steps in the DNA replication process: 1) Initiation 2) Unwind strands 3) Polymerize – add matched nucleotides to template 4) Clean up and connect We considered the replication of the circular chromosome of E. coli. A large body of work indicated that there is a single origin of replication and the forks move in a bi-directional manner. The isolation of mutants that perturb DNA replication has greatly contributed to the molecular details of how replication occurs. The single origin in E. coli is bound by the initiator protein, DnaA . The origin is called, oriC , and is a 250 bp region on the E. coli chromosome. The DnaA protein binds this sequence and wraps the DNA around itself to separate the DNA strands. DNA helicases are then loaded at the origin to facilitate unwinding of the helix. E. coli has lots of helicases, the major helicase that functions in DNA replication is encoded by the DnaB gene. Helicases use the energy from ATP hydrolysis to disrupting the hydrogen bonds between the bases in the double helix. These proteins look like rings around the DNA. Helicases are needed at initiation and throughout replication to continue to unwind the DNA as the replication fork moves. Once helicases melt the dsDNA, the resulting ssDNA needs to be coated by the single strand binding protein or SSB . This protein protects and stabilizes the ssDNA. The DNA is now accessible to the replication machinery. Because DNA polymerases cannot synthesize DNA de novo on the template, a special polymerase is needed to perform this function. This is the job of primase. Primase is a DNA- directed RNA polymerase that lays down a short RNA primer. The 3’-OH of the RNA primer is then utilized by DNA polymerase to extend the growing DNA chain. Because there are two strands of DNA, but DNA polymerase can only synthesis in the 5’ 3’ direction, there is a directionality to DNA synthesis, which is referred to as leading and lagging strand synthesis. The lagging strand uses the synthesis of Okazaki fragments to maintaining the directionality of
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Lecture13S10 - BIS101/Engebrecht Lecture 13 4/29/10 The...

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