The two replication forks move in opposite directions sequentially around the circular chromosome around(the(circular(chromosome( unique ori ! fully ! replicated ! The Phage Chromosome Three forms of DNA o Linear o H-bonded o Covalently bonded circular AT-rich and GC-rich regions used to demonstrate bidirectional replication Denaturation mapping Increase temperature or pH to denature genome Easier to break A-T bonds than G-C bonds because G-C have more hydrogen bonds By examining the positions of the branch point (Y-shaped structures) relative to the positions of the denaturing bubbles in a large number of Y-shaped replicative intermediates, Schnos and Inman demonstrated that both branch points are replication forks that move in opposite directions around the circular chromosome Key Points DNA replicates by a semiconservative mechanism: as the two complementary strands of a parental double helix
unwind and separate, each serves as template for the synthesis of a new complementary strand 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 10 6 nucleotide pairs and replicates as a single bidirectional replicon in approx. 40 minutes under a wide variety of growth conditions. The Supermegabigia coli chromosome contains approx. 8 x 10 7 nucleotide pairs and replicates at the same rate as E.coli How 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 10 6 nucleotide pairs in E. coli (or half the total nucleotide pairs).! Same principle applies to “ Supermegabigia# coli ” chromosome; it must traverse 4 x 10 7 nucleotide pairs.! 4 x 10 7 nucleotide pairs in “ Supermegabigia# coli ” ! 2 x 10 6 nucleotide pairs in E. coli ! ! = 20 times more nucleotide pairs in ! “ Supermegabigia# coli ” ! 40 minutes to replicate all the nucleotide pairs in E. coli x 20 ! ! = 800 minutes ! ! DNA Replication in Prokaryotes Must understand the process and the different proteins that enable this process Names of proteins involved How the process works Know how each protein works Don’t need to know structures of proteins Won’t be asked how many subunits
Many different DNA polymerases E.coli contains at least 5 different DNA polymerases: o DNA polymerase I –multiple functions (polymerase and nuclease) o DNA polymerase II – replication of damaged DNA o DNA polymerase III – multiple functions (polymerase and nuclease) o DNA polymerase IV – replication of damaged DNA o DNA polymerase V – replication of damaged DNA (dNMP) n + dNTP (dNMP) n+1 + PP i DNA template DNA dependent DNA polymerase DNA replication cannot occur de novo (from nothing) o Primer with a free OH group at its 3’ end o Template DNA to specify the sequence of the new DNA strand
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