7.4 DNA Replication Precisely Duplicates the Genetic MaterialDNA Sequences at Replication OriginsBacterial Replication OriginsEukaryote Replication OriginsReplication Initiation in BacteriaInitiation of ReplicationInitiation of Replication, continuedRNA Primers Are Needed for DNA ReplicationContinuous and Discontinuous Strand ReplicationLeading and Lagging Strand SynthesisRNA Primer Removal and Okazaki Fragment LigationSlide 12Slide 13Simultaneous Synthesis of Leading and Lagging StrandsThe Sliding ClampSlide 16Slide 17DNA ProofreadingEukaryotic DNA PolymerasesSlide 20TelomeresTelomeraseTelomeres, Aging, and CancerAbnormal Reactivation of Telomerase ActivitySlide 25The Polymerase Chain ReactionComponents of PCRThe Process of PCRSteps of PCRSlide 30Limitations of PCRSeparation of PCR ProductsVariable Number Tandem RepeatsSlide 34Slide 35Dideoxynucleotide DNA SequencingSanger SequencingThe Principle of Sanger SequencingSlide 39Slide 40Slide 41Slide 42Visualization of DNA SequenceSlide 44Automated DNA SequencingAutomated DNA Sequencing, continuedSlide 47Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach7.4 DNA Replication Precisely Duplicates the Genetic Material1•Replication is best studied in bacteria.•Though replication is very similar among Bacteria, Archaea, and Eukarya, the processes are not identical.•The enzymes and proteins involved are parts of large complex aggregations of proteins and enzymes called replisomes.•These assemble at each replication fork.Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated ApproachDNA Sequences at Replication Origins2•Replication origins have sequences that attract replication enzymes.•The origin of replication sequence of E. coli is called oriC, and it contains about 245 bp of A-T rich DNA.•The origin is divided into three 13-bp sequences follwed by four 9-bp sequences.Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated ApproachBacterial Replication Origins3•Replication origins of bacterial species have similar (conserved) but not identical sequences.•Comparison between species leads to identification of consensus sequences, the nucleotides found most often at each position of DNA in the conserved region.•The 13-mer and 9-mer sequences of oriC are conserved—they play an essential role in replication.Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated ApproachEukaryote Replication Origins4• Saccharomyces cerevisiae (yeast) has the most fully characterized origin-of –replication sequences.• The multiple origins of replication are called autonomously replicating sequences (ARS).• ARS organization and sequence is similar throughout the yeast genome.• Replication origins of other eukaryotes are less well characterized.Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated ApproachReplication Initiation in Bacteria5•Replication in E. coli requires that replication-initiating enzymes locate and bind to oriC consensus sequences.•Enzymes DnaA, DnaB, DnaC bind at oriC and initiate DNA replication.•DnaA binds first, bends the DNA, and breaks hydrogen bonds in the A-T rich sequences.Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated ApproachInitiation of Replication6•DnaA first binds the 9-mer sequences, bends the DNA, and breaks hydrogen bonds in the A-T rich sequences of the 13-mer region.•DnaB is a helicase that uses ATP energy to break hydrogen bonds of complementary bases to separate the strands and unwind the helix.•DnaB is carried to the DNA helix by DnaC.Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated ApproachInitiation of Replication, continued7•The unwound DNA strands are kept from re-annealing by single-stranded binding protein (SSB).•Unwinding of circular chromosomes will create torsional stress, potentially leading to supercoiled DNA.•Enzymes called topoisomerases catalyze controlled cleavage and rejoining of DNA that prevents overwinding.Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated ApproachRNA Primers Are Needed for DNA Replication8•DNA polymerase elongates DNA strands by adding nucleotides to the 3’ end of a pre-existing strand.•They cannot initiate DNA strand synthesis on their own.•RNA primers are needed; these are synthesized by a specialized RNA polymerase called primase.•Primase and some additional proteins join DnaA at oriC to form the primosome.Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated ApproachContinuous and Discontinuous Strand Replication9•In E. coli, daughter DNA strands are synthesized by the DNA polymerase III (pol III) holoenzyme.•Holoenzyme refers to a multiprotein complex in which a core enzymes is associated with the additional components needed for full function.•The replisome is found at each replication fork and contains two copies of pol III.Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated ApproachLeading and Lagging Strand Synthesis10•One copy of pol III synthesizes one daughter strand continuously in the same direction as fork progression, this is the leading strand.•The other copy of pol III elongates the daughter strand discontinuously, in the opposing direction to the fork progression, via short fragments (Okazaki fragments), this is the lagging strand.Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated ApproachRNA Primer Removal and Okazaki Fragment Ligation11•DNA polymerase I (pol I) uses two activities to complete replication:•Its 5 to 3 exonuclease activity removes the RNA primers.•Its 5 to 3 polymerase activity adds DNA nucleotides to the 3’ end of the DNA segment preceding the primer.•DNA ligase seals the gap between the resulting DNA segments (it does NOT add any bases though).Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach12Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach13Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated ApproachSimultaneous Synthesis of Leading and Lagging Strands14•Each replisome complex carries out replication of the leading and lagging strand simultaneously.•The DNA pol III holoenzyme contains 11 protein subunits, with the two pol III core polymerases each linked to a different copy of the tau (τ) protein.•The tau proteins