1. DNA replication Semi-conservative mechanism 1958, Meselson & Stahl
15N labeling experiment

2. Discovery of DNA structure
Rosalind Franklin ( ) Maurice Wilkins ( )
Francis Crick ( )
James Watson (1928-)
Discovery of DNA structure
1962 Nobel Prize

4. The substrates of DNA synthesis
dNTPs – dATP, dGTP, dCTP, dTTP
Direction: 5’-3’

6. 5’ ’ OH
T C A
ppp OH C
5’PPP +
T C A C
OH 3’
5’PPP
ppi

7. 3’ ’ ??? G
ppp OH
A T C G +
5’ ppp OH 3’
G A T C G
5’ ppp OH 3’

8. A T C G +
5’ ppp OH 3’ G
ppp OH G
5’ ppp
ppp OH 3’
A T C G
???

9. Proofreading??? + A T C G ppp OH ppp A p OH T C G pp p OH T C G T C G

10. Replicon is any piece of DNA which replicates as a single unit
Replicon is any piece of DNA which replicates as a single unit. It contains an origin and sometimes a terminus
Origin is the DNA sequence where a replicon initiates its replication.
Terminus is the DNA sequence where a replicon usually stops its replication

11. All prokaryotic chromosomes and many bacteriophage and viral DNA molecules are circular and comprise single replicons.
There is a single termination site roughly 180o opposite the unique origin.

12. The long, linear DNA molecules of eukaryotic chromosomes consist of mutiple regions, each with its own orgin.
A typical mammalian cell has replicons with a size range of kb. When replication forks from adjacent replication bubbles meet, they fuse to form the completely replicated DNA. No distinct termini are required

13. Semi-discontinuous replication
Experimental evidences
[3H] thymidine pulse-chase labeling experiment
1. Grow E. coli
2. Add [3H] thymidine in the medium for a few second, spin down and break the cell to stop labeling, analyze and find a large fraction of nascent DNA ( nt) = Okazaki fragments
3. Grow the cell in regular medium then analyze, the small fragments join into high molecular weight DNA = Ligation of the Okazaki fragments

15. Back

17. Bacterial DNA replication
Experimental systems
1. Purified DNA: smaller and simpler
bacteriophage and plasmid DNA molecules
(ΦX174, 5 Kb)
2. All the proteins and other factors for its
complete replications

18. Initiation: oriC Study system
the E. coli origin locus oriC is cloned into plasmids to produce more easily studied minichromosomes which behave like
E.coli chromosome.

19. 1. oriC contains four 9 bp binding sites for the initiator protein DnaA. Synthesis of DnaA is coupled to growth rate so that initiation of replication is also coupled to growth rate.
2. DnaA forms a complex of molecules, facilitating melting of three 13 bp AT-rich repeat sequence for DnaB binding.
3. DnaB is a helicase that use the energy of DNA hydrolysis to further melt the double-stranded DNA .
4. Ssb (single-stranded binding protein) coats the unwinded DNA.
5. DNA primase attaches to the DNA and synthesizes a short RNA primer for synthesis of the leading strand.
6. Primosome DnaB helicase and DNA primase

21. Unwinding Positive supercoiling: caused by removal of
helical turns at the replication fork.
Resolved by a type II topoisomerase called
DNA gyrase

23. Elongation DNA polymerase III holoenzyme
1. A dimer complex, one half synthesizing the leading strand and the other lagging strand.
2. Having two polymerases in a single complex ensures that both strands are synthesized at the same rate
3. Both polymerases contain an
α-subunit---polymerase
ε-subunit---3’ ’ proofreading exonuclease
β-subunit---clamp the polymerase to DNA
other subunits are different.

25. Other two enzymes during Elongation
Replisome
in vivo DNA polymerase holoenzyme dimer, primosome (helicase) are physically associated in a large complex to synthesize DNA at a rate of 900 bp/sec.
Other two enzymes during Elongation
1. Removal of RNA primer, and gap filling with DNA pol I
2. Ligation of Okazaki fragments are linked by DNA ligase.
Prokaryotic DNA replication

28. Termination and segregation
Terminus
containing several terminator sites (ter) approximately 180o opposite oriC.
Tus protein
ter binding protein, an inhibitor of the DnaB helicase
Topoisomerase IV
a type II DNA topoisomerase, function to unlink the interlinked daughter genomes.

31. Eukaryotic DNA replication
Experimental systems
1. Small animal viruses (simian virus 40, 5 kb) are good mammalian models for elongation (replication fork) but not for initiation.
2. Yeast (Saccharomyces cerevisiae): 14 Mb in 16 chromosomes, 400 replicons, much simpler than mammalian system and can serve as a model system
3. Cell-free extract prepared from Xenopus (frog) eggs containing high concentration of replication proteins and can support in vitro replication.

32. Cell cycle

33. Entry into the S-phase
Cyclins
CDKs (Cyclin-dependent protein kinases)

35. DNA Replication
DNA replication is semi-conservative, one strand serves as the template for the second strand.
Furthermore, DNA replication only occurs at a specific step in the cell cycle.
The following table describes the cell cycle for a hypothetical cell with a 24 hr cycle.
Stage Activity Duration
G1 Growth and increase in cell size 10 hr
S DNA synthesis hr
G2 Post-DNA synthesis hr
M Mitosis hr
DNA replication has two requirements that must be met:
1. DNA template
2. Free 3' -OH group

37. Origin and initiation
1. Clusters of about replicons initiate simultaneously at
defined times throughout S-phase
Early S-phase: euchromatin replication
Late S-phase: heterochromatin replication
Centromeric and telomeric DNA replicate last
2. Only initiate once per cell cycle
Licensing factor
required for initiation
inactivated after use
can only enter into nucleus when the nuclear envelope dissolves at mitosis

38. Electron Microscopy of replicating DNA reveals
replicating bubbles.

39. 3. Individual yeast replication origins (ARS) have been cloned into prokaryotic plasmids which allow these plasmids to replicate in yeast (an eukaryote).
ARSs autonomously replicating sequences
Minimal sequence 11 bp
[A/T]TTTAT[A/G]TTT[A/T] (TATA box)
4. ORC (origin recognition complex) binds to ARS, upon activation by CDKs, ORC will open the DNA for replication.

41. Elongation 1. Replication fork
- unwinding DNA from nucleosomes: 50 bp/sec
- need helicases and replication protein A (RP-A)
- new nucleosomes are assembled to DNA from a mixture
of old and newly synthesized histones after the fork passes

42. 2. Elongation Three different DNA polymerases are involved
1) DNA pol α contains primase activity and synthesizes RNA primers for the leading strands and each lagging strand fragments. Continues elongation with DNA but is replaced by the other two polymerases quickly.
2) DNA pol δ on the leading strand that replaces DNA pol α., can synthesize long DNA
3) DNA pol ε on the lagging strand that replaces DNA pol α., synthesized Okazaki fragments are very short (135 bp in SV40), reflecting the amount of DNA unwound from each nucleosome.

44. Nuclear matrix
1. A scaffold of insoluble protein fibers which acts as an organizational framework for nuclear processing, including DNA replication, transcription
2. Replication factories
containing all the replication
enzymes and DNA associated
with the replication forks
in replication
BudR labeling of DNA

45. Telomere replication

47. Telomerase
1. Contains a short RNA molecule as telomeric DNA synthesis template
2. Telomerase activity is repressed in the somatic cells of multicellular organism, resulting in a gradual shortening of the chromosomes with each cell generation, and ultimately cell death (related to cell aging)
3. The unlimited proliferative capacity of many cancer cells is associated with high telomerase activity.

48. cell dies or does not divide
Telomerase activity is repressed in somatic cells of multicelluar organisms resulting in a gradual shortening of the chromosome with each cell generation. As this shortening reaches informational DNA, the cells senesce and die.
When telomerase activity is repressed
informational DNA
cell division
cell dies or does not divide

51. Mutagenesis
Mutation
Permanent, heritable alterations in the base sequence of DNA
Reasons
1. Spontaneous errors in DNA replication or meiotic recombination
2. A consequence of the damaging effects of physical or chemical mutagens on DNA

52. Point mutation A singe base change: transition, transversion
The effects of point mutation
Phenotypic effects
Noncoding DNA
Nonregulatory DNA Silent mutation No
3rd position of a codon
Coding DNA altered AA Missense mutation Yes or No
Coding DNA stop codon Nonsense mutation Yes
Truncated protein

56. Insertions & deletions
The addition or loss of one or more bases in a DNA region
Frameshift mutations
The ORF of a protein encoded gene is changed so that the C-terminal side of the mutation is completely changed.
Genetic polymorphisms
Caused by accumulation of many silent and other nonlethal mutations

58. Replication fidelity Molecular mechanisms for the replication fidelity
Important for preserve the genetic information from one generation to the next, spontaneous errors in DNA replication is very rare, e.g. one error per 1010 base in E. coli.
Molecular mechanisms for the replication fidelity
1. DNA polymerase: Waston-Crick base pairing
2. 3’ 5’proofreading exonuclease.
3. RNA priming: proofreading the 5’end of the lagging strand
4. Mismatch repair

59. Mutagens Causing DNA damage that can be converted to mutations.
Physical mutagens
High-energy ionizing radiation
X-rays and γ-rays strand breaks and base/sugar destruction
Nonionizing radiation
UV light pyrimidine dimers
Chemical mutagens
Base analogs direct mutagenesis
Nitrous acid deaminates C to produce U
Alkylating agents
Arylating agents indirect-lesion mutagenesis
Intercalators: e.g. EB

60. Mutagenesis Direct mutagenesis
The molecular process in which the mutation is generated.
Note the great majority of lesions introduced by chemical and physical mutagens are repaired by one or more of the error-free DNA repair mechanisms before the lesions is encounter by a replication fork
Direct mutagenesis
The stable, unrepaired base with altered base pairing properties in the DNA is fixed to a mutation during DNA replication.

61. Indirect mutagenesis
The mutation is introduced as a result of an error-prone repair.
Translesion DNA synthesis
to maintain the DNA integrity but not the sequence accuracy
when damage occurs immediately ahead of an advancing fork, which is unsuitable for recombination repair, the daughter strand is synthesized regardless of the the base identity of the damaged sites of the parental DNA.

62. DNA damage and repair DNA lesions Oxidative Bulky adducts damage
1. Occurs under
normal condition
2. Increased by
ionizing radiation
physical mutagens
Bulky adducts
UV light
physical mutagens
Carcinogen
Chemical mutagens
Alkylation
Alkylating agents
Chemical mutagens

63. Biological effects of the unrepaired DNA lesions
Physical distortion of
the local DNA structure
Blocks replication
and/or transcription
Lethal
Altered chemistry of the bases
Allowed to Remain in the DNA
A mutation could become fixed by direct or indirect mutagenesis
Mutagenic

64. Spontaneous DNA lesions
1. Inherent chemical reactivity of the DNA
2. The presence of normal, reactive chemical species within the cell
- Deamination
C U
methylcytosine T
- Depurination
break of the glycosylic bond, non-coding lesion
- Depyrimidine

65. Oxidative damage
1. occurs under NORMAL conditions in all aerobic cells due to the presence of reactive oxygen species (ROS), such as superoxide, hydrogen peroxide, and the hydroxyl radicals (•OH).
2. The level of this damage can be INCREEASED by hydroxyl radicals from the radiolysis of H2O caused by ionizing radiation

67. Alkylation
1. Electrophilic chemicals adds alkyl groups to various positions on nucleic acids
2. Distinct from those methylated by normal methylating enzymes.
3. Typical alkylating agents:
MMS methylmethane sulfonate
EMS ethylmethane sulfonate
ENU ethylnitrosourea

69. Bulky adducts
1. DNA lesions that distort the double helix and cause localized denaturation, for example pyrimidine dimers
arylating agents adducts
2. These lesions disrupt the normal function of the DNA

73. DNA repair Photoreactivation
1. Monomerization of cyclobutane pyrimidine dimers by DNA photolyases in the presence of visible light
2. Direct reversal of a lesion and is error-free

75. Alkyltransferase
1. Removing the alkyl group from mutagenic O6-alkylguanine which can base-pair with T. The alkyl group is transferred to the protein itself and inactivate it.
2. Direct reversal of a lesion and is error-free
3. In E.coli, The response is adaptive because it is induced by low levels of alkylating agents and gives increased protection against the lethal and mutagenic effects of the high doses

77. Excision repair 1. Including nucleotide excision repair (NER)
base excision repair (BER)
2. Ubiquitous mechanism repairing a variety of lesions.
3. Error-free repair

78. Nucleotide excision repair (NER)
1. An endonuclease
cleaves DNA a precise
number of bases on
both sides of the lesions
(e.g. in E.coli, UvrABC
Endonulcease removes
pyrimidine dimers)
2. Excised lesion-DNA
fragment is removed
3. The gap is filled by
DNA polymerase I
and sealed by ligase

79. Base excision repair
(BER)

81. Mismatch repair A specialized form of excision repair which
deals with any base mispairs produced
during replication and which have escaped
proofreading

82. The parental strand is methylated at N6 position of all As in GATC sites, but methylation of the daughter
strand lag a few minutes after replication
MutH/MutS recognize the mismatched
base pair and the nearby GATC
DNA helicase II, SSB, exonuclease I
remove the DNA fragment including
the mismatch
DNA polymerase III & DNA
ligase fill in the gap

88. Essay questions
1. How to explain the mechanisms of semi-conservative replication and semi-discontinuous replication? How to verify them by experiments?
2. How about the differences between prokaryotic and eukaryotic DNA replication?
3. How about the main types of DNA damage? and the main repair mechanisms?

89. DNA recombination - Homologous recombination
- Site-specific recombination
- Transposition
An important reason for variable DNA sequences
among different populations of the same species

90. Homologous recombination
The exchange of homologous regions between two
DNA molecules
In diploid eukaryotes, it commonly occurs during meiosis
1. Homologous duplicated chromosomes line up in parallel in metaphase I.
2. The nonsister chromatids exchange equivalent sections by crossing over.

93. Crossing over

94. Haploid prokaryotes recombination
Occurs between the two homologous duplex
- between the replicated portions of a partially
duplicated DNA
- between the chromosomal DNA and acquired
“foreign” DNA, like plasmids or phages

97. Nick formation

98. RecA-ssDNA
filament

99. Recombination-based DNA repair

100. Site-specific recombination
1. Exchange of non-homologous but specific pieces of DNA
2. Mediated by proteins that recognize specific DNA sequences.

101. Bacteriophage λ insertion
1. λ -encoded integrase (Int): makes staggered cuts in
the specific sites
2. Int and IHF (integration host factor encoded by
bacteria): recombination and insertion
3. λ -encoded excisionase (XIS): excision of the
phage DNA

102. Antibody diversity
H and L are all encoded by three gene segments: V, D, J
V D J
Two heavy chains (L)
Two light chains (H)
Enormous number (>108) of different H and L gene sequences can be produced by such a recombination

103. Transposition 1. Requires no homology between sequences nor
site- specific
2. Relatively inefficient
3. Require Transposase encoded by the transposon

104. Transposons E. coli - IS elements/insertion sequence
1-2 kb, comprise a transposase gene flanked by a short inverted
terminal repeats
Tn transposon series
carry transposition elements and β-lactamase
(penicillin resistance)

105. Eukaryotic transposons
many are retrotransposons
Yeast Ty element encodes protein similar to RT
(reverse transcriptase)