Chapter 12 Outline

• 12.1 Genetic Information Must Be Accurately Copied Every Time a Cell Divides, 316

• 12.2 All DNA Replication Takes Place in a Semiconservative Manner, 316

• 12.3 The Replication of DNA Requires a Large Number of Enzymes and Proteins, 324

• 12.4 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:

• 14N common form; 15N rare heavy form

• E. coli were grown in a 15N media first, then transferred to 14N 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