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bubbles formed by 3 13 bp sequences that are all the same, rich in A:T bps, 4 9 bp sequences mediates protein binding to form the replication bubble- Requirements for DNA Synthesis:
Free 3’-OH group on the endTemplate strand to specify complementary bpsSubstrates: dNTP's (deoxynucleotide triphosphate)DNA polymeraseMg 2+- DNA Polymerase I:Covalent extension of DNA primer strand 5’-3’ direction (5’-3’ polymerase activity)Existing chain terminates at the 3’ end with deoxyguanylate DNA polymerase adds deoxythymidine to the 3’ end of the chain & releases pyrophosphateProof reads nascent DNA strand (3’-5’ exonuclease activity)Excision of RNA primers (5’-3’ exonuclease activity, occurs at same time as polymerase activity)-Leading Strand:3’-5’, DNA synthesis occurs continuously creating a strand that is 5’-3’- Lagging Strand:5’-3’, discontinuous elongation, requires RNA primers, forms okazaki fragments, DNA forms in overall 3’-5’ direction, but individual segments are replicated 5’-3’- DNA Ligase:Catalyzes closure of “nicks” in DNA molecules, missing phosphodiester bonds, not bases, requires ATP or NADLigase-AMP intermediate forms a phosphoester bond w/ 5’-phosphateNucleophilic attack by 3’-OH at the nick on the DNA- Formation of Replication Bubble in E. coli:DNA A protein binds to the 4 9 bp sequence in Ori CAdditional DNA A binds forming a complex w/ Ori C wrapped on the surface DNA B protein & DNA C protein join the initiation complex & produce a replication bubble - DNA Primase:Conduct DNA G geneCatalyzes synthesis of short RNA strands (10-60 nucleotides in prokaryotes, 10 in eukaryotes) complementary to template strands Provides the required 3’-OH for DNA polymerase to work - DNA Polymerase III:Adds deoxyribonucleotides to RNA primers, 5’-3’ polymerase activity Works continuously on leading strand, discontinuously on laggingStops extending Okazaki fragment once it bumps into the preceding Okazaki fragment- DNA Helicase:Unwinds DNA double helix
Uses ATP - Single Stranded Binding Proteins (SSB Proteins):Keeps unwound strand in extended form for replication w/out SSB proteins, DNA could renature or form hairpin structures- Topoisomerases:Catalyze transient breaks in DNA molecules, but use covalent linkages to themselves to hold on to cleaved molecules, use this energy to reseal the breaksTopoisomerase I: produces temporary single strand breaks or nicks in DNA, removes supercoils one at a time Topoisomerase II: produces transient double strand breaks in DNA, removes supercoils two at a time- DNA Primosome:Made of primase & helicaseInitiates formation of Okazaki fragments - Replisome:Complete replication apparatus moving along the DNA molecule at a replication fork