O primase adds new rna primers to the lagging strand

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o Primase adds new RNA primers to the lagging strand as it emerges from DnaB. o The gamma subunit (and other polypeptides) are associated with the lagging strand polymerase. Gamma is part of the clamp loader, a protein that puts the sliding clamp on to the lagging strand . o The lagging strand polymerase drops its old clamp and binds the newly loaded clamp as it begins a new Okazaki fragment. o DNA polymerase I is required to remove the RNA primer and replace it with DNA. o This leaves a nick in the DNA which is sealed by DNA ligase . o As the parental strands are unwound, positive supercoils accumulate ahead of the fork; these are relieved by the activity of gyrase .
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DNA replication is tightly controlled at initiation * The initiation of DNA synthesis occurs at a single site, oriC , in the E. coli chromosome. o CONCEPT: location at which DNA synthesis is initiated: the origin of replication . o The single replication origin of E. coli, oriC , has special sites that bind the regulatory protein DnaA . o The helicase, DnaB , is recruited by DnaA to help open up the DNA at the origin. o Primase binds the open DNA near the DnaB and DnaA complex and synthesizes short RNA primers. o This further opens the DNA and initiates the assembly of a replication complex at each of the two replication forks. o Origin DNA has a GATC+AT rich repeated sequence . o The AT rich property of the sequence may be important for opening the strands. o Negative supercoiling by gyrase may also contribute to opening. o The sequence GATC is methylated (on the N6 of the A) and this promotes binding of DnaA protein . o "old" DNA is methylated on both GATC strands, new strands are not methylated, thus recently replicated DNA is hemimethlyated . o the seqA protein binds hemimethylated DNA and sequesters it so that it is only slowly methylated. o The extra time provided by seqA prevents initiation from occurring rapidly multiple times in a row, and the cell can regulate initiation of replication.
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* DNA polymerases with special properties: o DNA polymerases from thermophilic archaea simplify the polymerase chain reaction. o Template independent Terminal deoxynucleotidyl Transferase. o Reverse transcriptase is a DNA polymerase that can use an exogenous RNA template. o Telomerase is a DNA polymerase that carries its own template.
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* Heat resistant polymerases and the polymerase chain reaction (PCR). o PCR provides for geometric amplification of a defined DNA sequence. o Template strand separation by heat denaturation of the DNA is the first step. o Annealing of the primers is the second step. o Extension of the primers is the third step. o Repeat the cycle by heating once more. o Whereas many proteins are irreversibly dentaured by heating to 95 degrees (necessary to denature DNA), DNA polymerase from thermophiles resists heat denaturation.
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