DNA Metabolism

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DNA Metabolism

• DNA replication processes by which copies of DNA
  molecules are faithfully made.
• DNA repair processes by which the integrity of
  DNA are maintained.
• DNA recombination processes by which the DNA
  sequences are rearranged.

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Map of the E. coli chromosome.
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DNA Replication Is Semiconservative.
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Replication Forks may Move Either
Unidirectionally or Bidirectionally
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Replication Begins at an Origin and Proceeds
Bidirectionally in Many Bacteria Such as E. coli.
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DNA synthesis is catalyzed by DNA polymerases in
the presence of (i) primer, (ii) template, (iii)
all 4 dNTP, and (iv) a divalent cathion such as
Mg.
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DNA Synthesis Cant be Continuously on Both
Strands (because the DNA duplex is antiparallel
and all DNA polymerases synthesize DNA in a 5 to
3 direction)
What is the source of primer used for lagging
strand synthesis?
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DNA Replication is Very Accurate

• Base selection by DNA polymerase is fairly
  accurate (about 1 error per 104)
• Proofreading by the 3 to 5 exonuclease
  associated with DNA polymerase improves the
  accuracy about 102 to 103-fold.
• Mismatch repair system repairs any mismatched
  base pairs remaining after replication and
  further improves the accuracy.

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An Example of Proofreading by the 3 to 5
Exonuclease of DNA Polymerase I of E. coli
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Large (Klenow) fragment of DNA polymerase I
retains polymerization and proofreading (3 to 5
exo)
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DNA polymerase I has 5 to 3 exonuclease and can
conduct Nick Translation
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Holoenzyme consists of two cores, two b subunits
and one g complex held together by a dimer of t.
So it is an asymmetric dimer.
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DNA polymerase III
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The two b subunits of PolIII form a circular
clamp that surrounds DNA
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DNA Replication requires many enzymes and protein
factors

• Helicases separation of DNA duplex.
• Topoisomerase relieves topological stress
• Single-strand DNA binding proteins stabilizes
  separated DNA strands.
• Primase synthesizes RNA primer.
• DNA Pol I removes RNA in Okazaki fragments and
  fills the gaps between Okazaki fragments.
• Ligase seals nicks.

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Replication of the E. coli chromosome

• Initiation.
• Elongation.
• Termination.

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Initiation begins at a fixed origin, called oriC,
which consists of 245 bp bearing DNA sequences
that are highly conserved among bacterial
replication origins.
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Model for initiation of replication at oriC.
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Proteins involved in Elongation of DNA
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Elongation Synthesis of Okazaki fragments
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Model for the synthesis of DNA on the leading and
lagging strands by the asymmetric dimer of PolIII
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Pol I can remove RNA primer and synthesize DNA to
fill the gap
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Termination When the two opposing forks meet in
a circular chromosome. Replication of the DNA
separating the opposing forks generated
catenanes, or interlinked circles.
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Termination sequences and Tus (termination
utilization substance) can arrest a replication
fork
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Replication in eukaryotic cells is more complex

• Contains many replicons.
• How is DNA replication initiated in each replicon
  is not well understood. Yeast cells appears to
  employ ARS (autonomously replicating sequences)
  and ORC (origin recognition complex) to initiate
  replication.
• More than one DNA polymerase are used to
  replicate DNA.
• End-replication problem of linear DNA.

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DNA Repair

• DNA damage may arise (i) spontaneously, (ii)
  environmental exposure to mutagens, or (iii)
  cellular metabolism.
• DNA damage may be classified as (I) strand
  breaks, (ii) base loss (AP site), (iii) base
  damages, (iv) adducts, (v) cross-links, (vi)
  sugar damages, (vii) DNA-protein cross links.
• DNA damage, if not repaired, may affect
  replication and transcription, leading to
  mutation or cell death.

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Ames test for mutagens (carcinogens)
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Methylataion and Mismatch Repair
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Model for Mismatch Repair
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Base-Excision Repair
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Nucleotide-Excision Repair in E. coli and Humans
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Direct Repair Photoreactivation by photolyase
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Alkylation of DNA by alkylating agents
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O6-methyl G, if not repaired, may produce a
mutation
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Direct Repair Reversal of O6 methyl G to G by
methyltransferase
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Direct repair of alkylated bases by AlkB.
Direct re
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Effect of DNA damage on replication (i) coding
lesions wont interfere with replication but may
produce mutation, (ii) non-coding lesions will
interfere with replication and may lead to
formation of daughter-strand gaps (DSG) or
double-strand breaks (DSB).
DSG and DSB may be repaired by recombination
process, to be discussed in the following section.
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DNA repair and cancer

• Defects in the genes encoding the proteins
  involved in nucleotide-excision repair, mismatch
  repair, and recombination repair have all been
  linked to human cancer.
• Examples are (i) xeroderma pigmentosum (or XP)
  patients with defects in nucleotide-excision
  repair, (ii) HNPCC (hereditary nonpoplyposis
  colon cancer) patients with defects in hMLH1 and
  hMSH2, and (3) breast cancer patients with
  inherited defects in BRCA1 and Brca2, which are
  known to interact with Rad 51 (the eukaryotic
  homolog of RecA) and therefore may have defective
  recombination repair.

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DNA Recombination

• Homologous recombination or generalized
  recombination.
• Site-specific recombinataion.
• Transposition.

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Pairing of homologous chromosomes and
crossing-over in meiosis.
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Recombination during meiosisis initiated by
double-strand breaks.
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Homologous recombination is catalyzed by enzymes

• The most well characterized recombination enzymes
  are derived from studies with E. coli cells.
• Presynapsis helicase and/or nuclease to generate
  single-strand DNA with 3-OH end (RecBCD).
• Synapsis joint molecule formation to generate
  Holliday juncture (RecA).
• Postsynapsis branch migration and resolution of
  Holliday juncture (RuvABC).

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Helicase and nuclease activities of the RecBCD
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RecA forms nucleoprotein filament on
single-strand DNA
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RecA promotes joint molecule formation and strand
exchange
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Model for DNA strand exchange mediated by RecA
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Models for recombinational DNA repair
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Models for recombinational DNA repair of stalled
replication fork
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Site-specific Recombination Bacteriophage lambda
integration in E. coli
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Effects pf site-specific recombination on DNA
structure
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A site-specific recombination reaction (eg.
catalyzed by Int of bacteriophage lambda)
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XerCD site-specific recombinataion system can
resolve dimer into monomer
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Immunoglobulin Genes Are Assembled by V(D)J
Recombination
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Mechanism of V(D)J Recombination
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Transposition

• Transposition is mediated by transposable
  elements, or transposons.
• Transposons of bacteria IS (insertion sequences)
  contains only sequences required for
  transposition and proteins (transposases) that
  promote the process. Complex transposons contain
  genes in addition to those needed for
  transposition.
• Transposition is characterized by duplication of
  direct repeats (5-9 bps) at target site.
• Transposition, in some instances, may be mediated
  through a RNA intermediate.

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Duplication of the DNA sequence at a target site
when a transposon is inserted
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Models for Direct and Replicative Transposition