DNA Replication and Recombination Benjamin A. Pierce GENETICS A Conceptual Approach SIXTH EDITION CHAPTER 12 DNA Replication and Recombination © 2017 W. H. Freeman and Company

The happy tree, Camptotheca acuminata, contains camptothecin, a substance used to treat cancer. Camptothecin inhibits cancer by blocking an important part of the replication machinery.

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

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

12.1 Three proposed models of replication are conservative replication, dispersive replication, and semiconservative replication.

Matthew Meselson and Franklin Stahl

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

12.2 Meselson and Stahl used equilibrium density gradient centrifugation to distinguish between heavy 15N DNA and lighter 14N DNA.

12.3 Meselson and Stahl demonstrated that DNA replication is semiconservative.

Concept Check 1 How many bands of DNA would be expected in Meselson and Stahl’s experiment after two rounds of conservative replication? 10

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 11

12.2 All DNA Replication Takes Place in a Semiconservative Manner 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

12. 4 Theta replication is a type of replication common in E 12.4 Theta replication is a type of replication common in E. coli and other organisms possessing circular DNA.

12.5 Rolling-circle replication takes place in some viruses and in the F factor of E. coli.

12.2 All DNA Replication Takes Place in a Semiconservative Manner Linear eukaryotic replication Eukaryotic cells Thousands of origins A typical replicon: ~ 200,000–300,000 bp in length Fig. 12.6

12.6 Linear DNA replication takes place in eukaryotic chromosomes.

12.2 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

12.7 New DNA is synthesized from deoxyribonucleoside triphosphates (dNTPs). The newly synthesized strand is complementary and antiparallel to the template strand; the two strands are held together by hydrogen bonds between the bases.

12.2 All DNA Replication Takes Place in a Semiconservative Manner Linear eukaryotic replication: Direction of replication DNA polymerase adds nucleotides only to the 3 end of growing strand Replication can only go from 5  3 Continuous and discontinuous replication Figs. 12.8 and 12.9

12.8 DNA synthesis takes place in opposite directions on the two DNA template strands. DNA replication at a single replication fork begins when a double-stranded DNA molecule unwinds to provide two single-strand templates.

12.2 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 fragments: discontinuously synthesized short DNA fragments forming the lagging strand

12.9 DNA synthesis is continuous on one template strand of DNA and discontinuous on the other.

Figure 12.10 The direction of synthesis in different models of replication.

Discontinuous replication is a result of which property of DNA? Concept Check 2 Discontinuous replication is a result of which property of DNA? Complementary bases Antiparallel nucleotide strands A charged phosphate group Five-carbon sugar 24

Discontinuous replication is a result of which property of DNA? Concept Check 2 Discontinuous replication is a result of which property of DNA? Complementary bases Antiparallel nucleotide strands A charged phosphate group Five-carbon sugar 25

Bacterial DNA replication 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 initiator protein (DnaA in E.coli) Unwinding: Initiator protein DNA helicase Single-strand-binding proteins (SSBs) DNA gyrase (topoisomerase)

12.11 E. coli DNA replication begins when initiator proteins bind to oriC, the origin of replication.

12.12 DNA helicase unwinds DNA by binding to the lagging-strand template at each replication fork and moving in the 5’ 3’ direction.

12.3 Bacterial Replication Requires a Large Number of Enzymes and Proteins Elongation: Primers: an existing group of RNA nucleotides with a 3-OH group to which a new nucleotide can be added. It is usually 10–12 nucleotides long. Primase: RNA polymerase

12.13 Primase synthesizes short stretches of RNA nucleotides, providing a 3’-OH group to which DNA polymerase can add DNA nucleotides.

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

Characteristics of DNA polymerases in E. coli TABLE 12.3 Characteristics of DNA polymerases in E. coli DNA Polymerase 5'  3' Polymerase Activity 3'  5' Exonuclease Activity 5'  3' Exonuclease Activity Function I Yes Removes and replaces primers II No DNA repair; restarts replication DNA halts synthesis III Elongates DNA IV DNA repair V DNA repair; translesion DNA synthesis

12.14 DNA ligase seals the break left by DNA polymerase I in the sugar–phosphate backbone.

Components required for replication in bacterial cells TABLE 12.4 Components required for replication in bacterial cells Component Function Initiator protein Binds to origin and separates strands of DNA to initiate replication DNA helicase Unwinds DNA at replication fork Single-strand-binding proteins Attach to single-stranded DNA and prevent secondary structures from forming DNA gyrase Moves ahead of the replication fork, making and resealing breaks in the double-helical DNA to release the torque that builds up as a result of unwinding at the replication fork DNA primase Synthesizes a short RNA primer to provide a 3'-OH group for the attachment of DNA nucleotides DNA polymerase III Elongates a new nucleotide strand from the 3' DNA polymerase I Removes RNA primers and replaces them with DNA DNA ligase Joins Okazaki fragments by sealing breaks in the sugar– phosphate backbone of newly synthesized DNA

12. 15 During DNA replication in E 12.15 During DNA replication in E. coli, the two units of DNA polymerase III are connected. The lagging-strand template forms a loop so that replication can take place on the two antiparallel DNA strands at the same time. Components of the replication machinery at the replication fork are shown at the top.

12.3 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

Concept Check 3 Which mechanism requires the ability to distinguish between newly synthesized and template strands of DNA? Nucleotide selection DNA proofreading Mismatch repair All of the above 37

Concept Check 3 Which mechanism requires the ability to distinguish between newly synthesized and template strands of DNA? Nucleotide selection DNA proofreading Mismatch repair All of the above 38

Eukaryotic DNA replication 12.4 Eukaryotic DNA Replication Is Similar to Bacterial Replication but Differs in Several Aspects 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

DNA polymerases in eukaryotic cells TABLE 12.5 DNA polymerases in eukaryotic cells DNA Polymerase 5' 3' Polymerase Activity 3'  5' Exonuclease Activity Cellular Function a (alpha) Yes No Initiation of nuclear DNA synthesis and DNA repair; has primase activity d (delta) Lagging-strand synthesis of nuclear DNA, DNA repair, and translesion DNA synthesis e (epsilon) Leading-strand synthesis g (gamma) Replication and repair of mitochondrial DNA j (zeta) Translesion DNA synthesis h (eta) u (theta) DNA repair i (iota) k (kappa) l (lambda) m (mu) s (sigma) Nuclear DNA replication (possibly), DNA repair, and sister-chromatid cohesion f (phi) Rev1 Note: The three polymerases listed at the top of the table are those that carry out nuclear DNA replication.

12.4 Eukaryotic DNA Replication Is Similar to Bacterial Replication but Differs in Several Aspects Eukaryotic DNA complexed to histone proteins in nucleosomes (Fig 12.16) 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. 16 Nucleosomes are quickly reassembled on newly synthesized DNA 12.16 Nucleosomes are quickly reassembled on newly synthesized DNA. This electron micrograph of eukaryotic DNA in the process of replication clearly shows that newly replicated DNA is already covered with nucleosomes (dark circles). [Victoria Foe.]

12.17 Experimental procedure for studying how nucleosomes dissociate and reassociate in the course of replication.

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. 12.18 Fig. 12.19

12.18 DNA synthesis at the ends of circular and linear chromosomes must differ.

12.19 The enzyme telomerase is responsible for the replication of chromosome ends.

Concept Check 4 What would be the result if an organism’s telomerase were mutated and nonfunctional? No DNA replication would take place. The DNA polymerase enzyme would stall the telomerase. Chromosomes would shorten each generation. RNA primers could not be removed. 47

Concept Check 4 What would be the result if an organism’s telomerase were mutated and nonfunctional? No DNA replication would take place. The DNA polymerase enzyme would stall the telomerase. Chromosomes would shorten each generation. RNA primers could not be removed. 48

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. 20 The Holliday model of homologous recombination 12.20 The Holliday model of homologous recombination. In this model, recombination takes place through a single-strand break in each DNA duplex, strand displacement, branch migration, and resolution of a single Holliday junction.

12. 21 The double-strand-break model of recombination 12.21 The double-strand-break model of recombination. In this model, recombination takes place through a double-strand break in one DNA duplex, strand displacement, DNA synthesis, and resolution of two Holliday junctions.

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

12.22 Gene conversion takes place through the repair of mismatched bases in heteroduplex DNA.