1. 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

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 model

5. 12.1 Three proposed models of replication are conservative replication, dispersive replication, and semiconservative replication.

6. Matthew Meselson and Franklin Stahl

7. 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

8. 12.2 Meselson and Stahl used equilibrium density gradient centrifugation to distinguish between heavy 15N DNA and lighter 14N DNA.

9. 12.3 Meselson and Stahl demonstrated that DNA replication is semiconservative.

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

11. 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. 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

13. 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.

14. 12.5 Rolling-circle replication takes place in some viruses and in the F factor of E. coli.

15. 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

16. 12.6 Linear DNA replication takes place in eukaryotic chromosomes.

17. 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

18. 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.

19. 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 and 12.9

20. 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.

21. 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

22. 12.9 DNA synthesis is continuous on one template strand of DNA and discontinuous on the other.

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

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. 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

26. 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)

27. 12.11 E. coli DNA replication begins when initiator proteins bind to oriC, the origin of replication.

28. 12.12 DNA helicase unwinds DNA by binding to the lagging-strand template at each replication fork and moving in the 5’ 3’ direction.

29. 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

30. 12.13 Primase synthesizes short stretches of RNA nucleotides, providing a 3’-OH group to which DNA polymerase can add DNA nucleotides.

31. 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. 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

33. 12.14 DNA ligase seals the break left by DNA polymerase I in the sugar–phosphate backbone.

34. 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

35. 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.

36. 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

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 37

38. 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

39. 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

40. 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.

41. 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

42. 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.]

43. 12.17 Experimental procedure for studying how nucleosomes dissociate and reassociate in the course of replication.

44. 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

45. 12.18 DNA synthesis at the ends of circular and linear chromosomes must differ.

46. 12.19 The enzyme telomerase is responsible for the replication of chromosome ends.

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. 47

48. 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

49. 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

50. 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.

51. 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.

52. 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

53. 12.22 Gene conversion takes place through the repair of mismatched bases in heteroduplex DNA.