The two replication forks move in opposite directions sequentially around the circular chromosome around(the(circular(chromosome(unique ori!fully !replicated!The Phage ChromosomeThree forms of DNAoLinearoH-bondedoCovalently bonded circularAT-rich and GC-rich regions used todemonstrate bidirectional replicationDenaturation mappingIncrease temperature or pH to denaturegenomeEasier to break A-T bonds than G-Cbonds because G-C have more hydrogenbondsBy examining the positions of the branch point(Y-shaped structures) relative to the positionsof the denaturing bubbles in a large number ofY-shaped replicative intermediates, Schnos andInman demonstrated that both branch pointsare replication forks that move in oppositedirections around the circular chromosomeKey Points DNA replicates by a semiconservativemechanism: as the two complementarystrands of a parental double helix
unwind and separate, each serves as template for the synthesis of a new complementarystrand The hydrogen bonding potentials of the bases in template strands specify complementary base sequences in the nascent DNA strands Replication is initiated at unique origins and usually proceeds bidirectionally from each origin Exercise:The Escherichia coli chromosome contains approx. 4 x 106nucleotide pairs and replicatesas a single bidirectional replicon in approx. 40 minutes under a wide variety of growth conditions.The Supermegabigia coli chromosome contains approx. 8 x 107nucleotide pairs and replicates at the same rate as E.coliHow long does it take to replicate the Supermeganigia coli?Is the answer:!!a) 80 minutes!!b) 120 minutes!!c) 800 minutes!!d) 2000 minutes!Because each chromosome replicates bidirectionally, each replication fork must traverse 2 x 106nucleotide pairs in E. coli (or half the total nucleotide pairs).!Same principle applies to “Supermegabigia#coli” chromosome; it must traverse 4 x 107nucleotide pairs.!4 x 107nucleotide pairs in “Supermegabigia#coli” !2 x 106nucleotide pairs in E. coli!!=20times more nucleotide pairs in !“Supermegabigia#coli” !40 minutes to replicate all the nucleotide pairs inE. coli x 20!!= 800 minutes!!DNA Replication in ProkaryotesMust understand the process and the different proteins that enable this processNames of proteins involvedHow the process worksKnow how each protein worksDon’t need to know structures of proteinsWon’t be asked how many subunits
Many different DNA polymerases E.coli contains at least 5 different DNA polymerases:oDNA polymerase I –multiple functions (polymerase and nuclease)oDNA polymerase II – replication of damaged DNAoDNA polymerase III – multiple functions (polymerase and nuclease)oDNA polymerase IV – replication of damaged DNAoDNA polymerase V – replication of damaged DNA(dNMP)n+ dNTP (dNMP)n+1+ PPi DNA template DNA dependent DNA polymerase DNA replication cannot occur de novo (from nothing)oPrimer with a free OH group at its 3’ end oTemplate DNA to specify the sequence of the new DNA strand