GENETICS A Conceptual Approach FIFTH EDITION GENETICS A Conceptual Approach FIFTH EDITION Benjamin A. Pierce CHAPTER 12 DNA Replication and Recombination © 2014 W. H. Freeman and Company

Replication has to be extremely accurate: –One error/million bp leads to 6400 mistakes every time a cell divides, which would be catastrophic. Replication also takes place at high speed. –E. coli replicates its DNA at a rate of 1000 nucleotides/second Genetic Information Must Be Accurately Copied Every Time a Cell Divides

12.2 All DNA Replication Takes Place in a Semiconservative Manner Proposed DNA Replication Models: –Conservative replication model –Dispersive replication model –Semiconservative replication

Meselson and Stahl’s Experiment: –Two isotopes of nitrogen: 14 N common form; 15 N rare heavy form E.coli were grown in a 15 N media first, then transferred to 14 N media Cultured E.coli were subjected to equilibrium density gradient centrifugation 12.2 All DNA Replication Takes Place in a Semiconservative Manner

Concept Check 1 How many bands of DNA would be expected in Meselson and Stahl’s experiment after two rounds of conservative replication?

Concept Check 1 How many bands of DNA would be expected in Meselson and Stahl’s experiment after two rounds of conservative replication? Two bands

Modes of Replication –Replicons: Units of replication. Replication origin –Theta replication: circular DNA, E. coli; single origin of replication forming a replication fork, and it is usually a bidirectional replication. –Rolling-circle replication: virus, F factor of E.coli; single origin of replication All DNA Replication Takes Place in a Semiconservative Manner

Linear eukaryotic replication –Eukaryotic cells –Thousands of origins –A typical replicon: ~ 200, ,000 bp in length. –Fig All DNA Replication Takes Place in a Semiconservative Manner

Linear eukaryotic replication: Requirements of replication –A template strand –Raw material: nucleotides –Enzymes and other proteins 12.2 All DNA Replication Takes Place in a Semiconservative Manner

Linear eukaryotic replication: Direction of Replication: –DNA polymerase add nucleotides only to the 3 end of growing strand. –The replication can only go from 5  3 –Continuous and discontinuous replication Figs and All DNA Replication Takes Place in a Semiconservative Manner

Linear eukaryotic replication: Direction of replication Leading strand: undergoes continuous replication. Lagging strand: undergoes discontinuous replication. Okazaki fragment: the discontinuously synthesized short DNA fragments forming the lagging strand All DNA Replication Takes Place in a Semiconservative Manner

Concept Check 2 Discontinuous replication is a result of which property of DNA? a.Complementary bases b.Antiparallel nucleotide strands c.A charged phosphate group d.Five-carbon sugar

Concept Check 2 Discontinuous replication is a result of which property of DNA? a.Complementary bases b.Antiparallel nucleotide strands c.A charged phosphate group d.Five-carbon sugar

12.3 Bacterial Replication Requires a Large Number of Enzymes and Proteins Bacterial DNA Replication –Initiation: 245 bp in the oriC. (single origin replicon) an initiation protein (DnaA in E.coli) –Unwinding: Initiator protein DNA helicase Single-strand-binding proteins (SSBs) DNA gyrase (topoisomerase)

Elongation: –Primers: an existing group of RNA nucleotides with a 3-OH group to which a new nucleotide can be added. It is usually nucleotides long. –Primase: RNA polymerase 12.3 Bacterial Replication Requires a Large Number of Enzymes and Proteins

Elongation: carried out by DNA polymerase III Removing RNA primer: DNA polymerase I ‒ Connecting nicks after RNA primers are removed: DNA ligase Termination: when replication fork meets or by termination protein Bacterial Replication Requires a Large Number of Enzymes and Proteins

The fidelity of DNA Replication Proofreading: DNA polymerase I: 3  5 exonuclease activity removes the incorrectly paired nucleotide. Mismatch repair: corrects errors after replication is complete Bacterial Replication Requires a Large Number of Enzymes and Proteins

Concept Check 3 Which mechanism requires the ability to distinguish between newly synthesized and template strands of DNA? a.Nucleotide selection b.DNA proofreading c.Mismatch repair d.All of the above

Concept Check 3 Which mechanism requires the ability to distinguish between newly synthesized and template strands of DNA? a.Nucleotide selection b.DNA proofreading c.Mismatch repair d.All of the above

Eukaryotic DNA Replication –Autonomously replicating sequences (ARSs) 100–120 bps –Origin-recognition complex (ORC) binds to ARSs to initiate DNA replication. –The licensing of DNA replication by the replication licensing factor MCM: Minichromosome maintenance –Eukaryotic DNA polymerase 12.4 Eukaryotic DNA Replication Is Similar to Bacterial Replication but Differs in Several Aspects

Eukaryotic DNA complexed to histone proteins in nucleosomes Nucleosomes reassembled quickly following replication Creation of nucleosomes requires: ‒ Disruption of original nucleosomes on the parental DNA ‒ Redistribution of preexisting histones on the new DNA ‒ The addition of newly synthesized histones to complete the formation of new nucleosomes 12.4 Eukaryotic DNA Replication Is Similar to Bacterial Replication but Differs in Several Aspects

The location of DNA replication within the nucleus: DNA polymerase is fixed in location and template RNA is threaded through it. Replication at the ends of chromosomes: ‒ Telomeres and telomerase. ‒ Fig ‒ Fig Eukaryotic DNA Replication Is Similar to Bacterial Replication but Differs in Several Aspects

Concept Check 4 What would be the result if an organism’s telomerase were mutated and nonfunctional? a.No DNA replication would take place. b.The DNA polymerase enzyme would stall the telomerase. c.Chromosomes would shorten each generation. d.RNA primers could not be removed.

Concept Check 4 What would be the result if an organism’s telomerase were mutated and nonfunctional? a.No DNA replication would take place. b.The DNA polymerase enzyme would stall the telomerase. c.Chromosomes would shorten each generation. d.RNA primers could not be removed.

12.5 Recombination Takes Place Through the Breakage, Alignment, and Repair of DNA Strands Homologous recombination: exchange is between homologous DNA molecules during crossing over. Holliday junction and single-strand break The double-strand break model of recombination

12.5 Recombination Takes Place Through the Breakage, Alignment, and Repair of DNA Strands Gene conversion: –process of nonreciprocal genetic exchange –produces abnormal ratios of gametes Gene conversion arises through heteroduplex formation