Patrick – Nucleic Acids
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big picture questions
knowing the diseases is important
B DNA is the most common, stable form; two right-handed helices
histones – made up of basic proteins (lysine and arginine)
o core histones: two of each type (4 types) per octamer
o linker histones: H1 (one per octamer)
o nucleosome: 1 unit of condensed DNA (including all core histones (8 total))
purines
o double ring
o A, G
o B-glycosidic linkage at N-9 position
pyrimidines
o single ring
o C, U, T
o B-glycosidic linkage at N-1 position
Canonical Watson-Crick base pairing
o G and C: 3 H bonds
o A and T: 2 H bonds
Base stacking due to hydrophobic interactions (as opposed to number of H bonds) is the
most important means of contributing to DNA stability
o GC base pairs exhibit more favorable base stacking, thus GC-rich DNA has a high
buoyant density
Chargaff’s Rule
o within a given duplex…
 #A = #T, and #G = #C
DNA written in 5`-3` direction (keep in mind when writing the antiparallel strand)
Major vs. minor groove
o different chemical environments
 helps control what protein or drug binds to a given DNA sequence
DNA replication induces supercoils which may interfere downstream proteins and
inhibit further replication
Single stranded DNA absorbs more light because the bases are more exposed
Interactions of drugs and proteins with DNA
o GC base pair exhibits a different environment than a CG base pair (also AT vs. TA)
o intercalating agents
 planar ring that fits between bases  DNA unwinding, lengthening
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e.g. daunorubicin (anticancer), EtBr (dye), actinomycin D (anticancer)
intercalation occurs between base stacking regions (ring structure)
sugar groups interact with the minor groove
these drugs are not specific when it comes to DNA binding, but they
exploit the excessive proliferation characteristic of cancer cells
o minor groove binding agents
 netropsin – AT sites; antimicrobial
 mitomycin C – CpG sites; anticancer
 Hoeschst 33258; dye
o DNA crosslinkers
 platinum-based drugs
 binds specific DNA sequences
 can’t distinguish between normal and cancer cell DNA
 distort duplex
 GG intrastrand
 GC interstrand
o Proteins
 protein alpha helix can fit in major groove
 TFs can H bond and recognize bases in major groove
 replication initiators, repair initiators
 histones – charge interaction with phosphodiester backbone
 Trp repressor – major groove interactions (binds specific DNA sequences)
 TATA binding protein (TBP) – minor groove
 Cro protein – interacts with both major and minor grooves as a dimer
Unusual base pairing
o Hoogsteen
 e.g. T-A-T, C-G-C
Patrick – DNA Replication1
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Tetraplex DNA – can form during DNA replication and recombination
o an example of Hoogsteen base pairing
o need runs of guanine (at least 3) separated by other bases, to stabilize this
structure
 abnormal H bonding
o will then block the necessary enzymes (helicases)
 Blooms
 growth retardation, sunlight, fertility, immunodeficiency, various
cancers, genome instability
 Werner’s
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recessive, rare, progeroid, cardiovascular, arthritis, various
cancers, growth retardation, thin + hardened skin, cataracts,
“bird-like” facial characteristics, genome + chromosome instability
Classes of DNA
o single copy: protein-coding
o middle-repetitive: tRNA, rRNA, pseudogenes
o repetitive
 SINEs
 LINEs – reverse transcriptase
 satellite
 Junk
Repeat expansion disorders
o Fragile X Syndrome
 from a CGG repeated motif in the 5 untranslated region of the FMR1
gene
 full mutation: >200 repeats  leads to lack of protein
o caused by…
 unequal cross-over
 amplification caused by defect in replication
 FMR1 gene
o an RNA binding protein; regulation of translation
o high expression in neurons
 X-linked dominant
 mental retardation
o Huntington’s Disease
 a polyglutamine repeat in the Huntingtin protein
 protein becomes polyglutaminated (toxic)
 autosomal dominant
 HTT gene
 expansion of CAG repeat
 severity increases with number of repeats
Restriction endonucleases
o often cleave DNA at palindromes
o defense against bacterial viruses
 methylation of host DNA
o restriction maps
 can use them to ID cleavage sites
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different distances between cleavage sites within genes of
different individuals – can look at polymorphic changes (i.e.
RFLPs) from one individual to the next  useful in parental
testing
e.g. sickle cell Hb gene
o Dde1 site is eliminated by a single nucleotide change
(AT)
 gel results will show only one heavier band (bc site
is absent, no cleavage occurs)
RNA
o folding patterns of large RNAs
 hairpin structures
 Hoogsteen
 Reverse Hoogsteen
 Base triples
 tertiary interactions
Enzymatic ddNTP sequencing
o 3` H is present and results in chain termination
Drugs: modified nucleosides
o acycloguanosine (acyclovir) – viral DNA infections; inhibits HSV DNA polymerase
o cytosine arabinoside – acute lymphocytic leukemia; inhibits topoisomerase I,
DNA and RNA polymerases
o azidothymidine – AIDS therapy; chain termination; 100-fold more specific for HIV
reverse transcriptase than DNA polymerases
dideoxy DNA sequencing
o large amount of template DNA (sequence in question) + dNTPs + smaller
amounts of ddNTPs
 in solution…random events will lead to termination of individual copies,
eventually yielding strands that end at each base of the template strand,
but with the complementary base
 each of these new, shorter strands is radioactively labeled, so
when run thru a gel, the fluorescing bands will separate based on
size and correspond to the antiparallel strand, as read from
lightest to heaviest strand
PCR
o known DNA sequence for primers, thermostable DNA polymerase, dNTPs, Mg2+,
DNA template, thermocycler
o fingerprinting and VNTRs
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DNA replication
o proposed methods
 conservative
 semiconservative
 dispersive
 replication would occur at about ~10 nucleotides at a time
o Meselson/Stahl Experiment
 if conservative, would only have seen heavy and light bands (no hybrid)
 if dispersive, would only have seen hybrid bands
Patrick – DNA Replication2
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DNA Chain extension
o in direction of 5` to 3`
 3` end of the template strand has hydroxyl group that participates in
nucleophilic attack on the phosphate of the dNTP precursor
o Prokaryotic Enzymes
 processivity
 DNA polymerase is a processive enzyme
o requires template and a primer (with a 3` OH group)
o DNA poly.I
 3`-5` exonuclease activity – for proofreading
 5`-3` exonuclease activity – primer removal
 moderately processive; fill in gaps; DNA repair
o DNA poly.III
 3`-5` exonuclease activity – for proofreading
 no 5`-3` exonuclease activity
 highly processive
 Primase = an RNA polymerase (not as processive as DNA
polymerase)
o synthesizes primer in 5`-3` direction
 DNApolyIII adds in 5`-3` direction
 DNA poly. I removes primer with 5`-3`
exonuclease activity and replaces with DNA
o finally, DNA ligase completes the
phosphodiester backbone (seals the
nick)
Replication
o initiation
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Topoisomerases
o topoisomerase II
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Eukaryotic DNA enzymes
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Termination of replication
o telomeres and telomerases
 telomerase synthesizes and extends telomeric DNA
DNA repair
o if there are faults in DNA repair, could increase the drive for generation of
mutations
o 1. Photoenzymatic repair
 upon DNA damage due to UV light
 cyclobutane dimer broken by photolyase
o 2. Base Excision Repair
 typically damage to a particular base
 could also involve uracil incorporation into DNA
 cleavage of glycosidic bond and removal of base
 then, need an endonuclease to break the phosphodiester
backbone, thus revealing a 3` OH
o (which enzyme possesses endonuclease activity?)
o 3. Nucleotide excision repair
 use of proteins to recognize a structural DNA abnormality (i.e. bulky DNA
damage)
 recruitment of DNA helicases and endonucleases (cleavage on
each side of damage strand)
o 4. Mismatch repair
 mismatched bases or potential “looped-out” bases (bases that aren’t
hybridized)
 parental vs. newly synthesized strand:
 (bc, don’t want to excise the parental, correct DNA)
 in bacteria  use of methylation to tag parental strand
 in humans  newly synthesized strand is recognized by unsealed
nicks in backbone
Know the major mechanisms of repair and the enzymes involved