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Nucleic Acid chemistry and technology

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Nucleic Acid chemistry and technology Implications of structural perturbations (hybridization) DNA synthesis and sequencing Non-enzymatic and enzymatic transformations – PowerPoint PPT presentation

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Title: Nucleic Acid chemistry and technology


1
Nucleic Acid chemistry and technology
  • Implications of structural perturbations
    (hybridization)
  • DNA synthesis and sequencing
  • Non-enzymatic and enzymatic transformations
  • Other functions

2
Nucleic acid structure can be disrupted
  • Similar to proteins, by heating, or change in pH,
    one can denature nucleic acid structures
  • Hydrogen bonds are broken, loss of base-stacking
    interactions cause strands of DNA double helix to
    separate
  • The strands can anneal once temperature or pH is
    returned to an appropriate temperature
  • dsDNA and ssDNA have distinct absorbance
    properties

3
Melting DNA is dependent upon GC content
4
Cyclical denaturation and renaturation of DNA
is basis of PCR
5
Hybridization is the key for microarray or gene
chip technologies
6
Cot analysis as a filter for repeat DNA sequences
7
Understanding the significance of DNA sequences
provides valuable insight into biology
  • Reactions
  • terminated by
  • dideoxy NTPs

8
Specific DNA sequences can be synthesized (i. e.
primers)
9
Alterations in DNA sequences
  • Purines and pyrimidines together with nucleotides
    undergo spontaneous alterations to their covalent
    structure
  • Deamination C ? U
  • Hydrolysis of bond between base and pentose
  • UV light can induce pyrimidine dimers
  • Alkylating agents
  • Oxidizing agents
  • Cells have several repair mechanisms, however,
    permanent alterations are mutations

10
Biosynthesis and degradation of nucleotides
  • Nucleotides are precursors of DNA and RNA,
    essential carriers of energy as ATP, GTP, NAD,
    FAD, CoA, etc., signaling mechanisms, and
    activated biosynthetic precursors.
  • Two pathways lead to nucleotide synthesis
  • de novo
  • salvage

11
de novo nucleotide synthesis
  • Appears identical among all organisms
  • Bases (guanine, etc.) are NOT intermediates in
    pathway
  • Purine rings not synthesized and attached to
    ribose, assembled on the ribose
  • Pyrimidine synthesized as orotate, attached to
    ribose phosphate and converted to nucleotides

12
Pyrimidines and purines share precursors
  • Phosphoribosyl pyrophosphate (PRPP) is a key
    intermediate for both (also involved in
    tryptophan and histidine synthesis also)
  • Amino acids are important precursors, glycine for
    purines, and aspartate for pyrimidines
  • Also, glutamine and aspartate serve as sources of
    amino groups in both purine and pyrimidine
    biosynthesis

13
PRPP serves as the foundation for purine
nucleotide biosynthesis
14
Three atoms from glycine are added to the new
amino group
15
This chain is extended by formate addition
16
An amine group is donated by glutamine
17
The FGAM ring is closed to form AIR
18
AIR is carboxylated to CAIR (unique because
doesnt use biotin)
19
A mutase rearranges the carboxylate
20
Aspartate donates an amino group in two steps to
form AICAR
21
Another formate group is donated, carried by THF
22
Ring closure forms Inosinate (IMP)
23
Summary of purine atom origins
24
IMP is converted to purine nucleotides
25
Regulation of purine biosynthesis
  • Three major feedback loops
  • Primary regualtion is AMP, GMP, and IMP
    inhibiting glutamine-PRPP amidotransferase (the
    first committed step)
  • Secondary regulation is inhibition of PRPP
    synthesis (where AMP and GMP act synergistically)
  • Also, regulated at bifurcation to AMP and GMP

26
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27
Pyrimidine biosynthesis from aspartate, PRPP, and
carbamoyl phosphate
  • Base (as orotate) is made first then attached to
    ribose 5-phosphate
  • Orotate synthesis begins with aspartate reacting
    with carbamoyl phosphate to form a product which
    is cyclized to Dihydroorotate
  • Dihydroorotate is oxidized to orotate, which
    reacts with PRPP
  • This product can undergo subsequent reactions to
    form UMP, UTP, and CTP

28
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29
Pyrimidine biosynthesis regulation
  • Mostly through the allosteric behavior of
    aspartate transcarbamoylase, which catalyzes the
    first step and is inhibited by CTP (inhibition
    can be prevented by ATP)

30
Nucleoside monophosphates are converted to
nucleoside triphosphates
  • AMP ? ADP (adenylate kinase)
  • ATP NMP ? ADP NDP (nucleoside monophosphate
    kinases)
  • Nucleoside diphosphate kinase converts nucleoside
    diphosphates to triphosphates (generally ATP is
    phosphate donor)

31
From these pathways, you note that
ribonucleotides are being generated
  • To get deoxyribonucleotides (precursors of DNA),
    the 2 carbon atom must be reduced
  • Accomplished by an interesting enzyme
    ribonucleotide reductase
  • A pair of hydrogen atoms originating from NADPH
    are passed to ribonucleotide reductase by either
    glutaredoxin or thioredoxin to generate an
    activated enzyme intermediate

32
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33
Ribonucleotide reductase catalytic mechanism
includes free radicals
34
Regulation of ribonucleotide reductase
  • Both activity and substrate specificity is
    modulated by binding of effector molecules
  • At one binding site
  • ATP activates enzyme
  • dATP inactivates enzyme
  • A second binding site
  • monitors substrate binding

35
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36
dTMP is generated from dUMP
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