Nucleotides and Nucleic Acids

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Dynamic Probes of Physical States in Live
Cells Presented by Dr. Brett Helms,
Molecular Foundry Lawrence Berkeley National
Laboratories Berkeley, California Part of
AMSECs MAD Seminar Series Materials Science
Seminar Monday, April 11th 515pm in CF110 /
Refreshments at 500pm in CF110
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Replication of Genetic Code

• Strand separation occurs first
• Each strand serves as a template
• for the synthesis of a new strand
• Synthesis is catalyzed by enzymes
• known as DNA polymerases
• Newly made DNA molecule has one
• daughter strand and one parent strand.

It has not escaped our notice that the specific
pairing we have postulated immediately suggests
a possible copying mechanism for the genetic
material Watson and Crick, in their
Nature paper,1953
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How would YOU go about determining the mechanism
of DNA replication?????

• What would a geneticist do?

What would a biochemist do?
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Figure 5-31 Action of DNA polymerases.
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Figure 5-32a Replication of duplex DNA in E. coli.
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Figure 5-32b Replication of duplex DNA in E. coli.
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Heres a computer modelhttp//www.youtube.com/wat
ch?v4jtmOZaIvS0
This is a pretty good outline http//www.youtube.
com/watch?vteV62zrm2P0NR1

• Overview of DNA and replication
• http//207.207.4.198/pub/flash/24/menu.swf

Another one with review questions http//www.wile
y.com/college/pratt/0471393878/student/animations/
dna_replication/index.html
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Molecular Mechanisms of Spontaneous Mutagenesis

• Deamination
• Very slow reactions
• Large number of residues
• The net effect is significant 100 C ? U
• events /day in a mammalian cell
• Depurination
• N-glycosidic bond is hydrolyzed
• Significant for purines 10,000 purines
• lost/day in a mammalian cell
• Cells have mechanisms to correct most of these
  modifications.

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Some well-characterized nonenzymatic reactions of
nucleotides.
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Some well-characterized nonenzymatic reactions of
nucleotides.
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Molecular Mechanisms of Oxidative and Chemical
Mutagenesis

• Oxidative damage
• Hydroxylation of guanine
• Mitochondrial DNA is most susceptible
• Chemical alkylation
• Methylation of guanine
• Cells have mechanisms to correct most of these
  modifications

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Molecular Mechanisms of Radiation-Induced
Mutagenesis

• UV light induces dimerization of pyrimidines,
  this may be the main mechanism for skin cancers
• Ionizing radiation (X-rays and ?-rays) causes
  ring opening and strand breaking. These are
  difficult to fix
• Cells can repair some of these modifications, but
  others cause mutations. Accumulation of
  mutations is linked to aging and carcinogenesis

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Formation of pyrimidine dimers induced by UV
light. (b) Formation of a cyclobutane pyrimidine
dimer introduces a bend or kink into the DNA
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http//www.nature.com/nature/journal/v421/n6921/fu
ll/nature01408.html
Review of DNA Repair Mechanisms

• Cute animations

Article to read for discussion on Friday
http//www.nature.com/nature/journal/v468/n7322/fu
ll/nature09428.html/base- excision-by-solvent-exp
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Nucleotide Excision Repair
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• DNA damage (black triangle) results in either
  repair or tolerance.
• a, During damage tolerance, damaged sites are
  recognized by the replication machinery before
  they can be repaired, resulting in an arrest that
  can be relieved by replicative bypass
  (translesion DNA synthesis).
• b, DNA repair involves the excision of bases and
  DNA synthesis (red wavy lines), which requires
  double-stranded DNA. Mispaired bases, usually
  generated by mistakes during DNA replication, are
  excised as single nucleotides during mismatch
  repair. A damaged base is excised as a single
  free base (base excision repair) or as an
  oligonucleotide fragment (nucleotide excision
  repair). Such fragments are generated by
  incisions flanking either side of the damaged
  base. Nucleotide excision repair can also
  transpire in some organisms by a distinct
  biochemical mechanism involving only a single
  incision next to a site of damage (unimodal
  incision).
• c, The cell has a network of complex signalling
  pathways that arrest the cell cycle and may
  ultimately lead to programmed cell death.

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Diseases

• colon cancer
• cellular ultraviolet sensitivity
• Werner syndrome (premature aging, retarded
  growth)
• Bloom syndrome (sunlight hypersensitivity)

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Damage of the double helix

• Single strand damage
• information is still backed up in the other
  strand
• Double strand damage
• no backup
• can cause the chromosome to break up

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Figure 30-51 Types and sites of chemical damage
to which DNA is normally susceptible in vivo.
Red, oxidation blue, hydrolysis green,
methylation.
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Endogenous and exogenous alkylating agents
(tobacco smoke, some anticancer drugs)
O6-alkylguanine has a different pattern of H-bond
donor and acceptor atoms than the parent guanine
base. As a result, it basepairs with T instead of
C, giving rise to G ? A transition after the
second round of replication
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Single strand repair

• Nucleotide excision repair
• a large multienzyme compound scans the DNA strand
  for anomalities
• upon detection a nuclease cuts the strand on both
  sides of the damage
• DNA helicase removes the oligonucleotide
• the gap is repaired by DNA polymerase and DNA
  ligase enzymes

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O6-alkylguanine DNA alkyltransferase
(AGT) Directly repaires alkylation damage
(O6-alkylguanines) by transferring the O6-alkyl
group from damaged guanine in DNA to a Cys
residue in the AGT active site in a
stoichiometric reaction. The protein is
inactivated via alkylation and undergoes
proteolytic degradation.
? AGT protein is highly conserved ?
helix-turn-helix DNA binding motif ? the
alkylated base is flipped out of the helix to
enter the hydrophobic alkyl-binding pocket of
the protein ? high metabolic cost for the cell is
outweighed by the need to maintain genetic
integrity
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Figure 30-52 The cyclobutylthymine dimer that
forms on UV irradiation of two adjacent thymine
residues on a DNA strand.
Photolyase repairs cyclobutane pyrimidine
dimers. Uses the energy of light to catalyze the
reversal of the cyclobutane bonds, producing
intact DNA. Not very important in mammals.
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Figure 30-55 The mechanism of nucleotide excision
repair (NER) of pyrimidine photodimers.
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Single strand repair

• Base excision repair
• A base-specific DNA glycosylase detects an
  altered base and removes it
• AP endonuclease and phosphodiesterase remove
  sugar phosphate
• DNA Polymerase fills and DNA ligase seals the
  nick

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Figure 30-56 Action of DNA glycosylases. These
enzymes hydrolyze the glycosidic bond of their
corresponding altered base (red) to yield an AP
site.
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Figure 30-57 X-Ray structure of human uracilDNA
glycosylase (UDG) in complex with a 10-bp DNA
containing a UG base pair.
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Base Excision Repair Used for repair of small
damaged bases in DNA (AP sites, oxidized,
deaminated, and methylated bases)
Several steps are involved a) modified base is
excised by N-glycosylase to give an abasic site
b) the abasic site is cleaved c) the resulting
single-nucleotide gap is filled by DNA Polymerase
d) DNA Ligase seals the nicks Example uracil
DNA glycosylase
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Figure 30-58The mechanism of mismatch repair in
E. coli.
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Mismatch Repair Mismatch Repair deals with
correcting mismatches of normal bases. Steps in
MMR Recognition of a mismatch Identification
of newly synthesized strand Removal of
mismatchGap repair by DNA Pol
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Figure 30-59 Regulation of the SOS response in E.
coli.
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Double strand repair

• Nonhomologous end-joining
• only in emergency situations
• two broken ends of DNA are joined together
• a couple of nucleotides are cut from both of the
  strands
• ligase joins the strands together

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Double strand repair

• Homologous end-joining
• damaged site is copied from the other chromosome
  by special recombination proteins

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DNA repair enzymes

• a lot of DNA damage -gt elevated levels of repair
  enzymes
• extreme change in cell's environment (heat, UV,
  radiation) activates genes that code DNA repair
  enzymes
• For an example, heat-shock proteins are produced
  in heat-shock response when being subjected to
  high temperatures.

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Cell Cycle and DNA repair

• Cell cycle is delayed if there is a lot of DNA
  damage.
• Repairing DNA as well as signals sent by damaged
  DNA delays progression of cell cycle.
• -gtensures that DNA damages are repaired before
  the cell divides

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Chapter 8 Summary
In this chapter, we learned about

• Function of nucleotides and nucleic acids
• Names and structures of common nucleotides
• Structural basis of DNA function
• Reversible denaturation of nucleic acids
• Chemical basis of mutagenesis