CHAPTER 25 DNA Metabolism

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CHAPTER 25 DNA Metabolism
Key topics

• DNA replication
• DNA repair
• DNA recombination

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What is DNA Metabolism?

• While functioning as a stable storage of genetic
  information, the structure of DNA is far from
  static
• A new copy of DNA is synthesized with high
  fidelity before each cell division
• Errors that arise during or after DNA synthesis
  are constantly checked for, and repairs are made
• Segments of DNA are rearranged either within a
  chromosome or between two DNA molecules giving
  offspring a novel DNA
• DNA metabolism consists of a set of enzyme
  catalyzed and tightly regulated processes that
  achieve these tasks

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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 Elongation Chemistry

• Parental DNA strand serves as a template
• Nucleotide triphosphates serve as substrates in
  strand synthesis
• Hydroxyl at the 3 end of growing chain makes a
  bond to the ?-phosphorus of nucleotide
• Pyrophosphate is a good leaving group

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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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PolIII consists of two cores, a clamp-loading
complex (g complex) consisting of t2 gdd, and
two additional proteins c and y. Holoenzyme is
PolIII plus b subunits.
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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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Assembly of a pre-replicative complex at a
eukaryotic replication origin
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The End Replication Problem of Linear DNA
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DNA Damages

• 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.

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DNA Repair and Mutations

• Chemical reactions and some physical processes
  constantly damage genomic DNA
• At the molecular level, damage usually involves
  changes in the structure of one of the strands
• Vast majority are corrected by repair systems
  using the other strand as a template
• Some base changes escape repair and the incorrect
  base serves as a template in replication
• The daughter DNA carries a changed sequence in
  both strands the DNA has been mutated
• Accumulation of mutations in eukaryotic cells is
  strongly correlated with cancer most carcinogens
  are also mutagens

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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.
• Case Files Case 11 (Breast cancer gene) and Case
  13 (Fragile X syndrome).

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DNA Recombination

• Segments of DNA can rearrange their location
• within a chromosome
• from one chromosome to another
• Such recombination is involved in many biological
  processes
• Repair of DNA
• Segregation of chromosomes during meiosis
• Enhancement of generic diversity
• In sexually reproducing organism, recombination
  and mutations are two driving forces of evolution
• Recombination of co-infecting viral genomes may
  enhance virulence and provide resistance to
  antivirals

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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 filaments are extended and disassembled in
the 5 to 3 direction
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Filament assembly is assisted by RecFOR and RecX
inhibits filament extension
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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 of stalled
replication fork
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Models for recombinational DNA repair
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Site-specific Recombination Bacteriophage lambda
integration in E. coli
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Effects of 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
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Replicative transposition is meidated by a
cointegrate intermediate.
Fig. 23.6
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Fig. 23.7