1. Chapter 5
DNA Replication, Repair, and Recombination

2. Outline of this chapter
The maintenance of DNA sequences
DNA replication mechanisms
The initiation and completion of DNA replication in chromosomes
DNA repair
General recombination
Site-specific recombination

3. The maintenance of DNA sequences
It is extremely important to keep the mutation rate low in all organisms.

4. Mutation and mutation rate
Mutation rate is the rate at which observable changes occur in DNA sequences.
Although human and E. coli are very different organisms, mutation rates of E. coli and human are approximately the same (10-9 per cell generation).

5. The estimation of mutation rate
The estimate of mutation rate can be done by looking at a gene that is not required for the survival of the organism. For example, the change of sequence of lactose-utilizing gene in E. coli grown under glucose-containing medium or the change of fibrinopeptide in mammals.

6. Mutation rate is often underestimated
Some mutation can happen undetected (‘silent’) if they don’t affect amino acid sequence or biological function of protein.
Also, mutations that dramatically affect biological function of the protein will vanish from population before they can be detected (‘preferential death’).

7. Unit evolutionary time
When we plotted the amino acid differences in a particular protein for several pairs of species against the time that has elapsed since the pair of species diverged from a common ancestor, the slope of this straight line is what we call the “unit evolutionary time”.

8. Unit evolutionary time
Unit evolutionary time reflects differences in the probability that an amino acid change will be harmful for each protein.
Some proteins like histone H4 are completely intolerant to mutation.

9. The effect of mutation rate to all organisms
Mutation rate has limit the number of essential proteins for any organisms to 60,000. If mutation rate is 10 percent higher (10-8 per cell generation), we will probably be fruit flies.

10. How is mutation rate being kept low enough?
Mutation rate is kept low enough by several mechanisms, including
- Proofreading of DNA polymerase
- DNA repair
At this moment we will discuss the proofreading of DNA polymerase

11. The first step of proofreading: correct nucleotide has a higher affinity for the moving polymerase

12. The second step: exonucleolytic proofreading
After an incorrect nucleotide is covalently added to the chain, DNA polymerase cannot continue the polymerization reaction until the incorect nucleotide is removed.

13. Exonucleolytic proofreading
This is because DNA polymerase has an absolute requirement for a base-paired 3’-OH end of a primer strand to continue its polymerization reaction.

14. Exonucleolytic proofreading
Most of the DNA polymerase has 3’-to-5’ exonuclease activity. This activity resides either in a separate domain of the same protein or in a separate subunit.

15. Exonucleolytic proofreading is probably why DNA is not synthesized from 3’-to-5’

16. DNA replication mechanisms
DNA replication is semiconservative and asymmetrical.

17. DNA replication is semiconservative
Because each of the two new DNA strands inherit one new strand and one old strand, we called this “semiconservative”.

18. DNA replication is semiconservative
1958, Matthew Meselson and Franklin Stahl

19. DNA replication is semiconservative

20. DNA replication is semiconservative

21. How is DNA being replicated?

22. The structure of DNA makes its replication asymmetrical
Because DNA is reverse and complementary (‘antiparallel’) and there is no 3’-to-5’ DNA polymerase, the replication of DNA must be progressed asymmetrically, with one strand moving faster (‘leading’) while the other moving slower (‘lagging’).

23. The lagging strand is lagging because it is synthesized discontinuously
Lagging strand DNA synthesis is delayed because it must wait for the leading strand to expose the template strand on which each Okazaki fragment is synthesized.

24. The Okazaki fragment
Okazaki fragments are the short DNA fragments produced during lagging strand DNA synthesis. They will be ligated together by ligase shortly after completion.
Prokaryotes like E. coli has Okazaki fragment of 1000~2000 nucleotides long while eukaryotes like us has shorter Okazaki fragments (100~200 nucleotides  nucleosomes?).

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When Okazaki used ligase mutant of bacteriophage T4 to perform the same experiment, he saw all the fragments are short fragments (Okazaki fragment).

26. The replication “fork”(fig. 5-6)
In the early 1960s John Cairns used radioactive DNA precursor to label replicating E. coli DNA. The results were like this picture.

27. weak label Strong label
J. Huberman and A. Tsai
Drosophila melanogaster
Strong label
Analyses carried out in the early 1960s on whole replicating chromosomes revealed a localized region of replication that moves progressively alone the parental DNA double helix, forming a Y-shaped structure.

28. DNA primase initiate new DNA synthesis
Because no DNA polymerase can initiate DNA synthesis anew, a different protein must initiate it.

29. DNA primase
DNA primase synthesize RNA primers to serve as initiation site for DNA polymerase.
In eukaryotes, this RNA primer is about 10 nucleotides long. In prokaryotes, it is about 12 nucleotide in length.

30. RNA primer is degraded after synthesis
After DNA is synthesized, RNA primer is being degraded and replaced by DNA (strand replacement synthesis).

31. DNA helicase opens up the DNA double helix
It is very difficult to make double stranded DNA unwind without any help.

32. Most of the DNA helicase is hexameric

33. Energy is required for the catalysis of DNA helicase
DNA helicases were first isolated as proteins that hydrolyzed ATP when they are bound to single strand DNA.

34. Single-strand DNA binding proteins (SSB) stabilize exposed single-stranded region

35. Without SSB, single stranded region will form short hairpins that hindering replication

36. SSB act cooperatively to bind single-stranded DNA

37. The processivity of DNA polymerase is enhanced by sliding clamp

38. Processivity
It means the tendency of a polymerase to stick with the replicating job once it starts.
When we said this polymerase is highly processive, meaning that once it starts replicating DNA, it won’t stop for a long time.

39. Sliding clamp The sliding clamp form a ring around the DNA helix.
One side of the ring binds to DNA polymerase while the whole ring slide freely along the DNA as the polymerase moves.
b clamp
PCNA

40. Sliding clamp is loaded by clamp loader, which is a five-protein complex

41. Clamp loading process requires ATP hydrolysis
Clamp loader interact with sliding clamp from the same side that DNA polymerase does.
Upon ATP hydrolysis, sliding clamp is loaded on to a primer-template junction.

42. Sliding clamp has different sides

43. Used clamp can be recycled by clamp loader

44. Clamp loader can be clamp unloader
The g-complex can unload b-clamp from replicated DNA.
The action of g-complex at this situation is also ATP-dependent.

46. In prokaryotes, the leading and lagging strand DNA replication machines are associated.

48. How is mutation rate kept low enough – mismatch repair
When an incorrect nucleotide was incorporated in synthesizing DNA strand undetected, it becomes a mismatch on the DNA.
Usually it will be caught by mismatch repair mechanism.

49. Mismatch repair
The most important thing for mismatch repair is the right strand cannot be repair according to the wrong strand sequence.
In prokaryotes like E. coli, newly synthesized strand is distinguished from the old strand by the methylation of A residues in the DNA of the sequence of GATC.

50. Mismatch repair in E. coli
MutS, MutL and MutH will recognize the methylated A on the GATC, then Mut H makes a nick 5’ of the mismatch….

51. Mismatch repair in E. coli
Exonuclease I, MutL, MutS, and helicase removes DNA 3’ of the nick, including the incorrect nucleotide. Because helicase is used in this reaction so ATP is required.

52. Mismatch repair in E. coli
DNA polymerase III fills the gap (with help from SSB) and DNA ligase seals the nick.

53. Mismatch repair in eukaryotes
While E. coli used methylated A in GATC sequences to distinguish old and new strands, eukaryotes like yeast and Drosophila do not have their DNA methylated.
However, newly synthesized DNA strands are known to be preferentially nicked (Okazaki fragments?), so this is used to distinguish old and new strands in eukaryotes.

54. Mismatch repair in eukaryotes
Eukaryotes also have MutL and MutS.
MutS binds to a mismatched base pair, while MutL scans the nearby DNA for a nick.

55. Mismatch repair in eukaryotes
Once a nick is found, MutL triggers the degradation of the nicked strand all the way back through the mismatch.

56. The winding problem that arises during DNA replication
As replication fork DNA unwinds, other parts of DNA are overwound.

57. The winding problem
For circular or extremely long DNA, the unwinding of replication results in overwinding of other parts of the DNA.

58. The winding problem
As the DNA is being replicated, the tension of the rest of DNA increases. If the tension is not relieved, DNA replication will come to a halt eventually because the tension is too big for helicase to unwind.

59. DNA topoisomerase
DNA topoisomerase can relieve the tension of the overwound DNA.
DNA topoisomerase can be viewed as a reversible nuclease that adds itself covalently to a DNA backbone phosphate. It breaks one or both strands of DNA, turn it around, ligate it back as it leaves.
There are two types of DNA topoisomerases, type I and II.

60. Topoisomerase I
Topoisomerase I produces a transient single-strand nick.

61. Topoisomerase I
This break allows the two sections of DNA helix on either side of the nick to rotate freely

62. Topoisomerase I
Afterwards, the nick is sealed by topoisomerase itself.
Topoisomerase I does not require ATP hydrolysis to complete its action.

63. Topoisomerase II Topoisomerase II breaks both strands of the DNA.
It is activated by sites on chromosomes where two double helices cross over each other.

66. The initiation and completion of DNA replication in chromosomes
How DNA replication is initiated and how cells carefully regulate this process

67. Replication origin
Replication origin is the position at which the DNA helix is first opened during replication.
Simple organisms have origins that contain DNA sequences several hundred nucleotide pairs in length.
Because A-T base pair is held together with fewer hydrogen bonds than G-C base pair, regions of DNA enriched in A-T pairs are typically found at replication origins.

68. Basic process of replication initiation

69. Bacterial chromosomes have a single replication origin
Bacteria like E. coli (4.6x106 bps) has a single origin of replication and the replication proceed bidirectionally with a speed approximately nt per second.

70. Replication initiation in bacteria

71. Replication initiation in bacteria

72. Replication initiation in bacteria
helicase

73. Replication initiation in bacteria

74. Replication initiation is tightly controlled
Initiation occurs only when sufficient nutrients are available for the bacterium to complete an entire round of replication.
Also, newly synthesized DNA with hemimethylated origins cannot be used for replication initiation.

75. Pulse-chase studies revealed how eukaryotes replicate their chromosomes

76. How eukaryotes replicate their chromosomes
Result suggested that multiple replication forks must proceed at the same time, since replication fork is estimated to travel at about 50 nt per second.

77. DNA replication in eukaryotes
Replication origins tend to be activated in clusters (replication units, origins).
New replication units seem to be activated at different times during the cell cycle until all of the DNA is replicated.
Individual origins are spaced at intervals of 30~300 kbp.
Replication also proceed bidirectionally from each replication origin.

78. DNA replication in eukaryotes
Eukaryotes only replicate their DNA during a specific part of the cell cycle (S phase).
Typical mammalian S phase is about 8 hours long (yeast: 40 minutes) while to finish replicating one origin takes only 1 hour. Therefore, the replication origins are not all activated simultaneously.

79. The timing of replication is related to the packing of the DNA in chromatin
Pulse-chase with bromodeoxyuridine (BrdU) found that different regions of each chromosome are replicated in a reproducible order during S phase.

80. The timing of replication is related to the packing of the DNA in chromatin
Observation done on female mammalian cell showed that heterochromatin tends to be replicated very late in the S phase. Later on, the observation on “housekeeping genes” also suggested that genes that are transcriptionally active (therefore least condensed) will be replicated first.
Chromosome condensation does not affect the speed of replication. It only affects the timing of replication initiation.

81. Yeast replication origin
ARS (autonomously replicating sequence) was found as yeast replication origin.

82. Yeast replication origin (ARS)
Each chromosome containing multiple ARS that are spaced 30 kb apart.

83. Minimum sequence of ARS
Minimum sequence requirement for a functional ARS was done by deletion studies on the original ARS sequences found.
The ORC-binding site is where the origin recognition complex bound.

85. How is DNA replication triggered?
ORC-origin form a stable complex throughout the cell cycle.
During G1 phase, a prerepicative complex (Mcm helicase and Cdc6 helicase loading factor) is assembled on each ORC.
S phase is triggered when a protein kinase is activated that assembles the rest of the replication machinery.

86. How does the mechanism ensure that a replication origin is used only once during each cell cycle?
After the rest of the replication machinery is assembled, Mcm helicase will start moving, forming two replication forks for each origin.
The protein kinase that triggers S phase will prevent all further assembly of the Mcm helicase into prereplicative complexes until it is inactivated (M phase).

87. Mammalian replication origin
Mammalian replication origin, such as b-globin gene cluster, contains several thousand nucleotides.
DNA sequences that are distant from origin affect the activity of replication origin (decondensing effect).

88. Mammalian replication origin
An ORC complex similar to yeast ORC is probably existed in mammals. Also, homologs of yeast Cdc6 and Mcm are found.
However, the binding sites for the ORC proteins seem to be less specific in mammals.

89. Eukaryotic DNA replication involves nucleosomes
Eukaryotic chromosomes are composed of the mixture of DNA and protein.
So chromosome duplication requires not only the DNA be replicated but also that new chromosomal proteins (histones) be synthesized and assembled onto the DNA behind each replication fork.

90. Histones remain associated with DNA after replication fork passes
By some unknown mechanism involving chromatin-remodeling proteins, the replication apparatus can pass through parental nucleosomes without displacing them from the DNA.
Both DNA helices inherit half of the old histones.

91. The structure of nucleosome

92. The synthesis of histones
Vertebrate cells have about 20 sets of each histone genes (H1, H2A, H2B, H3, and H4).
Histones are synthesized during S phase. Increased transcription and decreased mRNA degradation makes the level of histone mRNA to increase fiftyfold.
Histone mRNA is quickly degraded after DNA synthesis is stopped.

93. The assembly of nucleosome
Nucleosomes are assembled by chromatin assembly factor (CAF).
Newly synthesized histones H3 and H4 are rapidly acetylated on their N-terminal tails.

94. The assembly of nucleosome
The acetylated H3 and H4 will be deacetylated after they are assembled into chromatin.

95. The termination problem
Prokaryotes have circular DNA which will become entangled at the end of DNA replication.
Eukaryotes have linear DNA which will have insufficient space for lagging strand synthesis to start a new primer. The consequences of this is shorter and shorter chromosomes after generation.

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In E. coli the terminus region consists of 6
termination sites, named
TerA-TerF, and
proteins called
(terminus
utilization
substance) binds to it.
When replication forks enter the terminus region they pause, resulting two entangled daughter duplex.

97. catenane Type I topo Type II topo
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Type I topo
catenane
Type II topo

98. Telomere and telomerase
Eukaryotes have special nucleotide sequences at the ends of their chromosomes, which are incorporated into telomeres, and attract an enzyme called telomerase.
Telomere DNA sequences are similar in many organisms. In humans, this sequence is GGGTTA, extending about 10,000 nucleotides.

99. Telomerase
Telomerase synthesizes telomere DNA using an RNA template that is a component of the enzyme itself.

100. The 3’ end at each telomere is always slightly longer than the 5’ end.

101. The end of telomere DNA is protected
The protruding 3’ end has been shown to loop back to tuck the single-stranded terminus into the duplex DNA of the telomeric repeat sequence.

102. The “tucked” 3’ overhang of telomere

103. Telomere length is used as a counting device of cell age
Experiments showed that cells that proliferate indefinitely have homeostatic mechanisms that maintain the number of these repeats within a limited range.

104. Yeast cells, which can divide indefinitely, control the length of their telomeres within a limited range

105. Telomeres and replicative cell senescence
Human fibroblasts normally proliferate for about 60 cell divisions in culture, then it will cease to divide, this phenomenon is called replicative cell senescence.
According to a hypothesis, somatic cells like fibroblast has telomerases that are turned off, so each time a it divides, about nucleotides are lost.

106. Telomeres and replicative cell senescence
Eventually, descendent cells cease to divide because their chromosomes are defective.
This hypothesis was proven to be correct in fibroblast supplied with active fibroblast.
However, mouse with defective telomerase does not age prematurely. However, these mice do have a pronounced tendency to develop tumors (dyskeratosis congenita).