Mis paired base pairs are excluded Base Pairs Containing Tautomers o Tautomeric

Mis paired base pairs are excluded base pairs

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Mis-paired base pairs are excluded Base Pairs Containing Tautomers o Tautomeric base pairs result from chemical rearrangements of the bonds within a base Tautomeric shifts occur spontaneously Tautomeric bases can participate in forming alternative base pairs The geometry of some of these base pairs are compatible with the catalytic sites of DNA polymerases. Isomerization of the tautomeric base to a normal base results in a mismatched base pair (i.e. T=G – enol form slide 4). Proofreading o High-fidelity DNA polymerases have two active sites. 1) The catalytic site for DNA synthesis. 2) A 3’ → 5’ exonuclease site for removing mis-incorporated nucleotides o DNA pol I and III are high-fidelity enzymes. o Other errors in replication occur Eukaryotic DNA Polymerases o Key concept : Although the details vary, the overall process of replication in eukaryotes is very similar to replication in prokaryotes. o Eukaryotic cells Cells have many DNA polymerases (~15) with specialized functions “Replisome” carries out genome replication Other DNA polymerases are involved in a variety of functions, such as DNA repair DNA synthesis is semi-conservative and bidirectional The mechanism is both template - and primer-dependent DNA synthesis is always in the 5’ →3’ direction o continuous leading strand synthesis
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o discontinuous lagging strand Most DNA polymerases have both presynthetic error control and proofreading activity. Differences Unique to Eukaryotes o Eukaryotic chromosomes are linear and can be much longer than bacterial DNA molecules o Study of the architecture of genomic DNA polymerase “replisome” is ongoing Replisome has both DNA Pol and DNA Pol Primase is in a complex with DNA Pol Many of the proteins involved in eukaryotic replication are similar to those found in bacteria, but the nomenclature is different . o Mechanistic differences Replication initiation is very different Eukaryotic chromosomes have multiple origins of replication o Coordinate regulation of origins requires “licensing” Replication elongation is similar but not identical The rate of DNA synthesis is slower (max. ~50 nt/sec) Okazaki fragments are smaller (100-200 nt) o In humans, ~50 million are synthesized during one round of replication Completion and joining of Okazaki fragments is different Replication termination is different in the details need to address convergence of replication forks Replication of telomeres is unique to eukaryotes. Eukaryotic Replisome: a Work in Progress o Familiar proteins with new names: DNA pol is core polymerase for the leading strand (epsilon = leading) DNA pol is core polymerase for the lagging strand (del = lagging) DNA Pol -primase is a complex that contains primase for RNA primer synthesis and a separate DNA synthesis activity MCM = helicase RFC = clamp loader PCNA = clamp
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  • Spring '08
  • ALLISON
  • Biochemistry, dna polymerases

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