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

2. 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.

3. 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.

4. 12.2 All DNA Replication Takes Place in a Semiconservative Manner
Proposed DNA Replication Models:
Conservative replication model
Dispersive replication model
Semiconservative replication

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

6. 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 a 15N media first, then transferred to 14N media
Cultured E.coli were subjected to equilibrium density gradient centrifugation

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

8. Figure 12.3 Meselson and Stahl demonstrated the DNA replication is semiconservative.

9. Concept Check 1
How many bands of DNA would be expected in Meselson and Stahl’s experiment after two rounds of conservative replication? 9

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 10

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

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

14. 12.2 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. 12.6

15. Figure 12.6 Linear DNA replication takes place in eukaryotic chromosomes.

16. 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

17. Figure 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.

18. 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 12.9

19. Figure 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.

20. 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 fragment: the discontinuously synthesized short DNA fragments forming the lagging strand.

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

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

23. 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 23

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 24

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

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

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

28. 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 nucleotides long.
Primase: RNA polymerase

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

30. 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.

32. Figure 12.14 DNA ligase seals the break left by DNA polymerase I in the sugar-phosphate backbone.

34. 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.

35. 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 35

36. 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 36

37. 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

39. 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

40. 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

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

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

43. 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. 43

44. 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. 44

45. 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

46. 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.

47. 12. 20 The double-strand-break model of recombination
12.20 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.

48. Gene conversion arises through heteroduplex formation
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

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