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The function of DNA is information transfer and storage. 1. DNA is copied to more DNA in DNA replication 2. Gene expression i.e. Transcription- synthesis of RNA from only one strand of a double stranded DNA helix
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The function of DNA is information transfer and storage 1. DNA is copied to more DNA in DNA replication 2. Gene expression i.e. Transcription- synthesis of RNA from only one strand of a double stranded DNA helix Translation- ribosome mediated synthesis of a polypeptide from a messenger RNA molecule
DNA replication A polymerisation reaction with the typical 3 phases • initiation, • elongation, • termination
Initiation • occurs at Ori C (origin of chrm replication) • a 245 bp region with 2 conserved sequences • 3 tandem 13bp repeats • 4 copies of a 9 bp sequence
Dna A protein molecules with bound ATP bind to 9 bp repeats • Facilitated by HU protein • The 3 x 13 bp repeats are sequencially denatured to give the open complex • A complex formed by Dna B and Dna C bind to the melted region, Dna C is released • In the presence of SSB protein and Dna gyrase the Dna B helicase unwinds the DNA in preparation for priming and DNA synthesis
Elongation • Leading and lagging strand synthesis • Parental DNA is unwound (helicases) and topological stress removed (gyrases/topoisomerases) • Separated strand stabilised by SSB • Leading strand • Primase (DnaG protein) synthesises a short RNA primer at origin • DNA pol III builds complimentary strand from ds primer • continuous process • Lagging • Primase synthesizes many RNA primers along lagging strand • Each primer is extended by DNA pol III • Synthesis proceeds in 5' to 3' direction i.e. the direction opposite to the fork movement • Synthesis is discontinuous in the form of multiple Okazaki fragments
lagging continued.......... • Synthesis continues until fragment extends as far as the primer of the previously added Okazaki sequence • Note both strands are synthesised by a single asymetric dimer of DNA pol III that moves in the direction of the replication fork • This is achieved in the case of the lagging strand by the DNA looping around the part of the dimer DNA pol III
DnaB (helicase) and Dna G (primase) together form the primosome • 1000 nucleotides of new DNA added per second to each strand • RNA primers are removed (exonuclease activity) and replaced with DNA (polymerase) by DNA pol I and the remaining nick is sealled by a ligase using NAD as a cofactor
Termination • Eventually the 2 relication forks of E.coli meet at a terminus region containing many copies of a 20 bp sequence called Ter • These sequences are binding sites for a protein called Terminal utilization substance (Tus) • Tus-Ter complex arrests the replication fork in one direction
The other replication fork halts when they meet. • the few hundred base pairs in between the protein complexes are replicated by an as yet unknown mechanism resulting in 2 interlinked circular chromosomes • separation requires Topoisomerase IV • separate chrm are then segregated at cell division
Proteins at the E coli replication Fork SSB binds to ssDNA and stabilizes it DnaB protein (helicase) DNA unwinding; primosome constituent Dna G protein (Primase) RNA primer synthesis; primosome constituent DNA pol III New strand elongation DNA pol I Excision of primers and filling of gaps DNA ligase Ligation DNA gyrase supercoiling (DNA topoisomerase II)
DNA polymerases DNA pol I DNA pol II DNA pol III Mol.wt. (Daltons) 103,000 88,000 900,000 polymerizatn rate 16-20 7 250-1000 (nucleotides/second) 3' to 5' exonuclease activity yes yes yes 5' to 3‘ exonuclease activity yes no no Functions proof reading and repair replicatn