Chapter 12 Outline 12.1 Genetic Information Must Be Accurately Copied Every Time a Cell Divides, All DNA Replication Takes Place in a Semiconservative Manner, The Replication of DNA Requires a Large Number of Enzymes and Proteins, Recombination Takes Place Through the Breakage, Alignment, and Repair of DNA Strands, 335

12.1 Genetic Information Must Be Accurately Copied Every Time a Cell Divides Replication has to be extremely accurate: 1 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.

12.2 All DNA Replication Takes Place in a Semiconservative Manner

Conservative replication model Dispersive replication model Semiconservative replication Proposed DNA Replication Models

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. Meselson and Stahl’s Experiment

Replicons: units of replication Replication origin Theta replication: circular DNA, E. coli; single origin of replication forming a replication fork, usually a bidirectional replication Rolling-circle replication: virus, F factor of E. coli; single origin of replication Modes of Replication

Eukaryotic cells; thousands of origins; a typical replicon: 200,000 ~ 300,000 bp in length Linear Eukaryotic Replication

Requirements of replication: A template strand Raw material: nucleotides Enzymes and other proteins Linear Eukaryotic Replication

Direction of replication: DNA polymerase add nucleotides only to the 3′ end of a growing strand. The replication can only go 5′  3′. 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 Linear Eukaryotic Replication

12.3 The Replication of DNA Requires a Large Number of Enzymes and Proteins

Bacterial DNA Replication Initiation: 245 bp in the oriC (single origin replicon); an initiation protein Unwinding of DNA is performed by Helicase. Gyrase removes supercoiling ahead of the replication fork. Single stranded DNA is prevented from annealing by single stranded binding proteins. Primers: an existing group of RNA nucleotides with a 3′- OH group to which a new nucleotide can be added; usually 10 ~ 12 nucleotides long Primase: RNA polymerase

Bacterial DNA Replication Elongation: carried out by DNA polymerase III Removing RNA primer: DNA polymerase I DNA ligase: connecting nicks after RNA primers are removed Termination: when a replication fork meets or by termination protein

Bacterial DNA Replication The fidelity of DNA replication Proofreading: DNA polymerase I: 3′  5′ exonuclease activity removes the incorrectly paired nucleotide. Mismatch repair: correcting errors after replication is complete

Eukaryotic DNA Replication Eukaryotic DNA polymerase DNA polymerase  acts like Primase to initiate DNA polymerase  - replicates lagging strand DNA polymerase  - replicates leading strand

Eukaryotic DNA Replication Replication at the ends of chromosomes: Telomeres and telomerase