DNA replication

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DNA replication
Semi-conservative mechanism
1958, Meselson Stahl 15N labeling experiment
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Rosalind Franklin (1920-1958)
Maurice Wilkins (1916-2004)
Francis Crick (1916-2004) James Watson (1928-)
Discovery of DNA structure 1962
Nobel Prize
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• The substrates of DNA synthesis
• dNTPs dATP, dGTP, dCTP, dTTP
• Direction 5-3

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5 3

5PPP
5PPP
ppi
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3 5 ???
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???
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Proofreading???
pp
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• 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

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• 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.

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• The long, linear DNA molecules of eukaryotic
  chromosomes consist of mutiple regions, each with
  its own orgin.
• A typical mammalian cell has 50000-100000
  replicons with a size range of 40-200 kb. When
  replication forks from adjacent replication
  bubbles meet, they fuse to form the completely
  replicated DNA. No distinct termini are required

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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 (1000-2000 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

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Back
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Bacterial DNA replication

• Experimental systems
• 1. Purified DNA smaller and simpler
• bacteriophage and plasmid DNA molecules
• (FX174, 5 Kb)
• 2. All the proteins and other factors for its
• complete replications

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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.

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• 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 30-40 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

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

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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
• a-subunit---polymerase
• e-subunit---3 5 proofreading
  exonuclease
• ß-subunit---clamp the polymerase to DNA
• other subunits are different.

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• 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
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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.

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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.

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• Cell cycle

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

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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 8 hr G2 Post-DNA synthesis 5
hr M Mitosis 1 hr DNA replication
has two requirements that must be met 1. DNA
template 2. Free 3' -OH group
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Origin and initiation

• 1. Clusters of about 20-50 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

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Electron Microscopy of replicating DNA
reveals replicating bubbles.
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• 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/TTTTATA/GTTTA/T (TATA box)
• 4. ORC (origin recognition complex) binds to ARS,
  upon activation by CDKs, ORC will open the DNA
  for replication.

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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

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• 2. Elongation
• Three different DNA polymerases are involved
• 1) DNA pol a 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 d on the leading strand that replaces
  DNA pol a., can synthesize long DNA
• 3) DNA pol e on the lagging strand that replaces
  DNA pol a., synthesized Okazaki fragments are
  very short (135 bp in SV40), reflecting the
  amount of DNA unwound from each nucleosome.

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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
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Telomere replication
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• 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.

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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
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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

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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

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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

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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 5proofreading exonuclease.
• 3. RNA priming proofreading the 5end of the
  lagging strand
• 4. Mismatch repair

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Mutagens

• Causing DNA damage that can be converted to
  mutations.
• Physical mutagens
• High-energy ionizing radiation
• X-rays and ?-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

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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.

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• 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.

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DNA damage and repair

• DNA lesions

Oxidative 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
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• 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
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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

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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

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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

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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

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• 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

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• 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

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

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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

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Base excision repair (BER)
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Mismatch repair

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

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• 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
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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?

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DNA recombination

• - Homologous recombination
• - Site-specific recombination
• - Transposition

An important reason for variable DNA
sequences among different populations of the same
species
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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.

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Crossing over
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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

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Nick formation
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RecA-ssDNA filament
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Recombination-based DNA repair
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Site-specific recombination

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

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Bacteriophage ? insertion

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

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Antibody diversity

• H and L are all encoded by three gene segments
  V, D, J

V
D J Two heavy chains (L)
250 15 5 Two light chains (H)
250 4
Enormous number (gt108) of different H and L gene
sequences can be produced by such a recombination
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Transposition
1. Requires no homology between sequences nor
site- specific 2. Relatively inefficient 3.
Require Transposase encoded by the transposon
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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 ß-lactamase
• (penicillin resistance)

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

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