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DNA replication

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DNA replication Semi-conservative mechanism 1958, Meselson & Stahl 15N labeling experiment The substrates of DNA synthesis dNTPs dATP, dGTP, dCTP, dTTP Direction ... – PowerPoint PPT presentation

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Title: DNA replication


1
DNA replication
Semi-conservative mechanism
1958, Meselson Stahl 15N labeling experiment
2
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

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 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

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 (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

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.

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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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