Dr. Chesnokov
September 16, 2008
Fundamentals I
11:00-12:00 pm
Slide 1: Outline
• How Is DNA Replicated?
• What Are the Properties of DNA Polymerases?
• How Is DNA Replicated in Eukaryotic Cells?
• How Are the Ends of Chromosomes Replicated?
• How Are RNA Genomes Replicated?
• How Is the Genetic Information Shuffled by Genetic Recombination?
• Can DNA Be Repaired?
• What Is the Molecular Basis of Mutation?
Slide 2: How are RNA Genomes Replicated?
• Many viruses have genomes composed of RNA
• DNA containing viruses would replicate the same as DNA in prokaryotic and eukaryotic cells…the
major features are the same
• Can viral RNA serve as a template for DNA synthesis?
• What enzyme could mediate such process?
Slide 3: Another way to Make DNA
 RNA-Directed DNA Polymerase
• 1964: Howard Temin notices that DNA synthesis inhibitors prevent infection of cells in culture by
RNA tumor viruses.
• Temin predicts that DNA is an intermediate in RNA tumor virus replication
• 1970: Temin and David Baltimore (separately) discover the RNA-directed DNA polymerase - aka
"reverse transcriptase"
Slide 4: Reverse Transcriptase
 All RNA tumor viruses contain a reverse transcriptase
 Primer required for synthesis of an RNA containing genome, but it is a strange one - a tRNA molecule
that the virus captures from the host cells.
 RT transcribes the RNA template into a complementary DNA (cDNA) to form a DNA:RNA hybrid
Slide 5: Reverse Transcriptase Activities
 Three enzyme activities for reverse transcriptase which is…
o RNA-directed DNA polymerase
o RNase H activity - degrades RNA in the DNA:RNA hybrids
o DNA-directed DNA polymerase - which makes a DNA duplex after RNase H activity destroys
the viral genome, the RNA strand
Slide 6: AZT Structure
 The structures of AZT (3¢-azido-2¢,3¢-dideoxythymidine).
 The first approved drug used for treatment of AIDS disease; codes for an RNA containing virus
 This compound serves as a substrate analog that binds to HIV reverse transcriptase
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This nucleoside was the first approved drug for treatment of AIDS. AZT is phosphorylated in vivo to
give AZTTP (AZT 5¢-triphosphate), a substrate analog that binds to HIV reverse transcriptase, HIV
reverse transcriptase incorporates AZTTP into growing DNA chains in place of dTTP. Incorporated
AZTMP blocks further chain elongation because its 3¢-azido group cannot form a phosphodiester
bond with an incoming nucleotide.
Host cell DNA polymerases have little affinity for AZTTP, but have some affinity- that’s why so many
of these drugs have side effects
Slide 7: How Is the Genetic Information Shuffled by Genetic Recombination?
• During this process, genetic recombination rearranges genetic information, creating new
associations
• Recombination involving similar DNA sequences is called homologous recombination
• Homologous recombination is achieved by the process of general recombination
• General recombination requires the breakage and reunion of DNA strands
• These are the major players: The proteins responsible include RecA, RecBCD, RuvA, RuvB, & RuvC
Slide 8: How Is the Genetic Information Shuffled by Genetic Recombination?
• Nonhomologous recombination – when very different nucleotide sequences recombine, occurs at
low frequency in genome.
• Transposition – enzymatic insertion of transposon (mobile segment of DNA) can occur.
• Nonhomologous recombination and transposition play significant evolutionary role
Slide 9: Meselson and Weigle’s Exeperiment
 Experiment that discovered that DNA can be rearranged.
 Demonstrated that a physical exchange of chromosome parts actually occurs during recombination.
 Infected bacterial cells with a so called “heavy” phage, which was originally grown on a substrate
containing heavy DNA precursors.
 The co-infected bacteria with phage containing “light” genome and tried to separate the progeny of
the phages on a density gradient and thus predicted phages containing heavy genome were
separated first
 Phages containing light genome were much higher during separation but surprisingly they found a
number of phages that contained both heavy and light genomes which were produced during
recombination process.
 Density-labeled, “heavy” phage) , (ABC), were used to co-infect bacteria along with ”light” (abc)
phage. The progeny from the infection were collected and subjected to CsCl density gradient
centrifugation.
 Parental-type heavy (ABC) and light (abc) phage were well separated in the gradient, but
recombinant phage particles (ABc,Abc, aBc,aBC, and so on ) were distributed diffusely between the
two parental bands because they contained chromosomes constituted from fragments of both
“heavy” and “light” DNA.
 These recombinant chromosomes formed by breakage and reunion of parental “heavy” and “light”
chromosomes.
Slide 10: Mechanism of Recombination
• General recombination: any pair of homologous DNA segments can be used as substrates
• In 1964, Robin Holliday proposed a model involving single-stranded nicks at homologous sites
• Duplex unwinding, strand invasion and ligation create a Holliday junction
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Slide 11: Holliday Model for homologous recombination
 Summarized in this cartoon here on the slide
 For the recombination to happen---(A) Two homologous DNA duplexes are aligned – synapsis.
(B) Recombination begins with the introduction of single-stranded nicks at homologous sites on two
chromosomes shown here
(C) Strand invasion occurs through partial unwinding and base-pairing with the intact strand in the
other duplex
(D) Free ends from different duplexes are ligated resulting in cross-stranded intermediate – Holliday
junction
(E) Branches can migrate by unwinding and rewinding of two duplexes
Slide 12: Holliday Model for homologous recombination
 At the end, Branch migration (E) results in strand exchange. Another pair of nicks must be
introduced to resolve Holliday junction into two DNA duplex molecules. Nicks take place either at E
an W (- strands) or at N and S (+ strands) resulting in “patch” or “splice” recombinant
heteroduplexes.
Slide 13: The Enzymology
 Includes several enzymes and the most important ones are summarized here:
• RecBCD initiates recombination in E.coli
• RecA forms nucleoprotein filament for strand invasion and homologous pairing
• RuvA, RuvB, RuvC drive branch migration and help to resolve the Holliday junction into
recombination products
• Eukaryotic systems are probably similar, because homologous proteins with similar functions have
been identified in eukaryotes.
Slide 14: Model of RecBCD-dependent initiation of recombination
 On the next few slides, this concept has been summarized in several cartoons.
 This is a model of RecBCD dependent-initiation of recombination
 RecBCD consists of three subunits and has both helicase and nuclease activities.
 x site – recombinational “hotspot” (5-GCTGGTGG-3), more than 1000 in E.coli.
Slide 15: Model of RecBCD-dependent initiation of recombination
 RecBCD binds to a duplex DNA end, and its helicase activity begins to unwind the DNA double helix.
 “Rabbit ears” of ssDNA loop out from RecBCD helicase because the rate of DNA unwinding exceeds
the rate of ssDNA release by RecBCD.
Slide 16: Model of RecBCD-dependent initiation of recombination
 ssDNA are coated by ssDNA binding protein and occasionally by RecA.
 As RecBCD moves along DNA until it encounters the x site.
 As it unwinds the DNA, SSB ( and some RecA) bind to the single-stranded regions; the RecBCD
endonuclease activity randomly cleaves the ssDNA, showing a greater tendency to cut the 3’terminal strand rather that the 5’-terminal strand.
Slide 17: Model of RecBCD-dependent initiation of recombination
 As soon as it encounters the x site, it cuts the DNA just below the 3’ end of the x site.
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When RecBCD encounters a properly oriented x site, the 3’-terminal strand is cleaved just below the
3’-end of x.
Slide 18: Model of RecBCD-dependent initiation of recombination
 At this point, ssDNA binding protein is displaced and the strand is coated by RecA protein which can
initiate strand invasion.
 (d) RecBCD now directs the binding of RecA to the 3’-terminal strand, as RecBCD endonuclease
activity now acts more often on the 5’-terminal strand. (e) A nucleoprotein filament consisting of
RecA-coated 3’-strand ssDNA is formed. This nucleoprotein filament is capable of homologous
pairing with a dsDNA and strand invasion.
Slide 19: The RecA Protein—recombinase.
 Several features of RecA protein which are summarized below.
 38 kD enzyme that catalyzes ATP-dependent DNA strand exchange, leading to formation of Holliday
junction
 RecA forms a helical filament with a groove to accommodate DNA
 RecA:ssDNA complex binds dsDNA at secondary site and searches for regions homologous with the
bound ssDNA, then forms the desired duplex
Slide 20: Structure of RecA, a 352-residue, 38 kD protein
 This is based on the crystal structure of RecA monomer with incorporated ATP.
 This is a structure of actually RecA filament and there 4 turns of helical filament and has about 6
RecA monomers and one monomer is shown in RED.
 Ribbon diagram of the RecA monomer. Note the ADP bound at the site near helices C and D.
 (b) RecA filament. Four turns of a helical filament that has six RecA monomers per turn. A RecA
monomer is highlighted in red. RecA filament can bind multiple DNA strands!
Slide 21: Model for homolgous recombination as promoted by RecA enzyme
 RecA protein (and SSBinding protein) aid strand invasion of the 3’-ssDNA into a homologous DNA
duplex,
 forming a D-loop.
 The D-loop strand, that has been displaced by strand invasion, pairs with its complementary strand
in the original duplex to form a Holiday junction as strand invasion continues.
Slide 22: Resolving Holliday Junction
 Ruv proteins play an important role and are summarized here along with a cartoon.
• Ruv proteins resolve the junction into recombination products
• RuvA and RuvB act as a helicase that dissociates the RecA filament and catalyzes branch migration
• RuvC is an endonuclease that binds at the junction and cuts pairs of DNA strands of similar polarity.
Both splice and patch recombinants can be produced.
Slide 23: Model for resolving Holliday junction
 (left) RuvA tetramer fits snugly within the Holliday junction point.
 (center) RuvB hexameric rings assemble on opposite sides of DNA heteroduplexes and act as motors
to promote branch migration by driving the passage of the DNA duplexes through themselves.
 (right) RuvC resolvase binds to the Holliday junction and cuts it by its nuclease activity.
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Slide 24: Knockout Mice: A Method to Investiage the Essentiality of a Gene based on homologous
recombination
 This method is very popular in the lab for creating so called knock-out mice. If you want to check the
essentially of a certain gene, you can create in vitro gene disrupted by, for example, neo gene, which
stands for neomysin which is an antibiotic.
 Cultures expressing neomysin can be identified in cell culture.
 Insertion of neomysin actually destroys the gene of interest and when inserted in these cells, this
particular culture can incorporation into genome and during homologous recombination can replace
the original gene.
 After a couple of generation you can get a homozygous mouse carrying only the disrupted copy of
the gene independent on the essentially of the gene—you can study it’s manifestations in the cell in
live animal.
Slide 25: Transposons
 Moblie elements of genome.
 In 1950, Barbara McClintock showed that activator genes in corn could move freely about the
genome.
 This was at first viewed as heresy
 Molecular biologists in the late 1970s confirmed what McClintock discovered
 She received a MacArthur Award in 1981 and a Nobel Prize in 1983
Slide 26: Transposons
 The process of transposon incorporation is shown here in this cartoon.
 Transposon is usually flanked by inverted nucleotide repeats at its termini and the size of the inverted
repeats can be up to 15 bp.
 The typical transposon has inverted nucleotide-sequence repeats at its termini, represented here as
the 12-bp sequence ACGTACGTACGT
 (a). Transposon acts at a target sequence (shown here as the sequence CATGC) within host DNA by
creating a staggered cut- approximately 6 bp in host DNA
 (b) whose protruding single-stranded ends are then ligated to the transposon
 (c). The gaps at the target site are then filled in, and the filled-in strands are ligated
 (d). Transposon insertion thus generates direct repeats of the target site in the host DNA, and these
direct repeats flank the inserted transposon.
Slide 27: Transgenic animals are animals carrying foreign genes
 This is another useful technique which arose from recombination and arrangement processes that
also resulted in the creation of transgenic animals where a gene of interest can be incorporated into
the egg of the mouse, incorporate into DNA, excised, and be incorporated into live animals which
can give birth and produce copy of the alien gene.
 In this case, rat growth hormone was inserted into the mouse, which results in mice twice the size as
usual.
Slide 28: DNA Repair
 Now I am going to speak about DNA repair.
 This process has a fundamental difference from RNA, protein, lipid, because all those others can be
replaced, but DNA must be preserved.
 Repair processes are found mostly for genetic materials in the cells.
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These cells require a means for repair of missing, altered or incorrect bases, or bulges due to
insertion or deletion, UV-induced alteration, strand breaks or cross-links
There are two principal mechanisms: mismatch repair and methods for reversing chemical damage
Slide 29: Mismatch Repair corrects errors introduced during DNA replication
• So mismatch repair actual corrects errors introduced in DNA replication.
• Mismatch repair systems scan DNA duplexes for mismatched bases, excise the mispaired region and
replace it
• An example is a Methyl-directed pathway of bacteria
• Since methylation occurs only after replication, repair proteins (MutS, MutH, MutL) identify
methylated strand as parent, (which is unchanged to serve as a template) remove mismatched
bases on the other strand and replace them
• So in the book that we are using for this class, mismatch repair wasn’t really described well so I will
just give you this Wikipedia reference where it is described in more details and is up to date:
http://en.wikipedia.org/wiki/Mismatch_repair
Slide 30: Reversing Chemical Damage (excision repair)
 Okay reversing chemical damage happens through excision repairs.
• For example, Pyrimidine dimers can
• In excision repair: DNA glycosylase removes damaged base, creating an "AP site"
• AP endonuclease cleaves backbone, exonuclease removes several residues around damaged
residue and gap is repaired by DNA polymerase and DNA ligase
Slide 31: UV radiation image
 I just want to show that this is what happens during UV irradation; it results in dimerization of
adjacent thymine bases.
 A cyclobutyl ring is formed between those carbon bases and the enzymes that actually cut this
ring can restore the actual thymine bases. This is called photolyase.
Slide 32: Base excision repair.
• This process is illustrated on this slide. Where you can see a damaged base in black excised
from the sugar/phosphate backbone by DNA glycoslyase, creating a so called AP site.
• Then an endonuclease severs the DNA strand and excision endonuclease removes the AP site
and several surrounding nucleotides.
• DNA Pol I and DNA ligases repairs again.
Slide 33: What is the molecular basis of mutation?
 Okay, what is the molecular basis of mutation?
• Point mutations usually arise by inappropriate base-pairing
• Mutations can be caused by base analogs
• Chemical mutagens react with bases in DNA
• Insertions and deletions result in frame shift mutations
Slide 34: Types of mutations
 Here I summarize several types of mutations which include:
o Large chromosomal deletions
o Translocations is swapping of chromosomal segments
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Point mutations:
 -Transition - Pu replaced by Pu or Py-Py
 -Transversion – Py replaced by Pu or Pu-Py
-Mis-sense mutation
 1 base substituted for another
-Frame shift - Alters the reading frame of the gene must be in protein coding region
insertion or deletion which results in a NON functional protein
Slide 35: Image: example of point mutations
 So here you can see the result of point mutations due to base mispairings in which occurs in unusual
circumstances in the cells.
 For example, the rare imino tautomer of adenine base pairs cytosine rather than thymine. Here is
the normal A-T base pair. In the case where a hydrogen atom is transferred from this N to this, it
can result in cytosine as the progeny from AT to GC base pair.
 Also, adenine in the syn confirmation can pair with G instead of T. I didn’t mention this yesterday
but bases and nucleotides can exist in both syn and anticonformation.
 So, what is it when you have a base in this position and a sugar here, the base can rotate in both
directions. Usually anti conformation is favored, but sometimes the base can flip. Bases in the syn
conformation, such as adenine, can interact with different bases but this occurs in unusual
circumstances.
 T and C form a base pair by H-bonding interactions mediated by a water molecule as shown here.
Then again, these circumstances are very rare and not usual.
Slide 36: Image - aminopurine
 This is an example of 2-Aminopurine (adenine analog) normally base-pairs with T but may also pair
with cytosine through a single hydrogen bond, resulting in a base substitution.
Slide 37: Image- base analog
 This is another example of a base analog.
 Oxidative deamination of adenine in DNA yields hypoxanthine, which base-pairs with cytosine,
resulting in an A-T to G-C transition.
Slide 38: Examples of Chemical mutagens
And here I listed a number of chemical mutagens with their formal mechanism of action. I am not going
to talk about all of them; I will just mention that some of them are actually very useful in lab studies as
very potent mutations. For example, labs that are using fruit flies can use nitrosoamines to induce
mutation is D. melanogaster.
 Ethylmethane sulfonate (EMS) can also be used to induce mutation and based on a new phenotype
can be identified as being involved in certain aspects of the cell cycle.
Slide 39: Frame shift image
 This is an example of a frame shift where you have a sequence of DNA and the corresponding amino
acids that correspond to certain condons in the DNA.
 Addition of guanine changes the protein sequence from the one here to completely something else.
 This results in a non function protein.
met phe gln gln phe
ATG TTT CAG CAA TTT
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met val ser ala
ile
ATG GTT TCA GCA ATT T
Slide 40: Diseases of DNA repair
 Here is list of disease caused by mistakes in DNA repair. They include Bloom syndrome, Fanconi
Anemia and many other diseases.
 I won’t go into detail about this.
Shows Movie: I want to show you a little movie – chromosome #11. Plays video.
Slide 41: Review
 I have several review slides. Usually we have 2 questions per 1 hour of lecture so I will go through
what might appear.
• Nucleotides and Nucleic Acids. There might be a question about the structure and functions of
nucleotide. You don’t need to know the exact formula but each nucleotide consists of a sugar, a
nitrogenous base and phosphate.
• Important discoveries which helped solving DNA structure – including Chargaff rule, X-ray
crystalography data, and the model by Watson and Crick.
• Major features of the DNA double helix. States that the strands in DNA run in an anti parallel
direction, the base pairing underlies the formation of the double helix.
Slide 42: Review
• Also concerning the structure of DNA. DNA can exist in 3 possible conformations (secondary
structures) called ABZs of DNA structure
• A is for DNA which is dehydrated and not usually found in vivo
• B is the right handed DNA helix which is most commonly found in cells.
• Z form occurs in the GC rich regions of DNA and is actually left handed.
Know primary, secondary and tertiary structure of DNA.
• Primary structure is essential a sequence of nucleotides.
• Secondary is the DNA helix
• Tertiary structure of DNA involves super coils that can be induced by DNA topoismerases.
Know the major features of DNA replication that are common for all cells. This includes unwinding of
DNA, DNA replication is bidirectional, semi discontinuous, and semi conservative.
Slide 43: Review
 Slide 44: DNA recombination and repair
• Enzymology of DNA recombination – know major enzymes important for DNA replication
- DNA pol – synthesizes the new strands
- DNA helicase – unwinds the helix
 Know differences in prokaryotic and eukaryotic replication. Basic features are the same, but there
are more proteins involved in eukaryotic DNA replications and several DNA pol. Those proteins are
more complex.
- Example, initiator protein in bacterial DNA consists of one subunit. In eukaryotes, its origin of
replication complex has 6 subunits.
 DNA recombination and repair – enzymes involved in DNA recombination and resolving Holladay
junction.
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Major types of DNA recombination and repair such as general recombination and mismatch
repair and excision repair.
Molecular basis of mutations – just don’t go into details or memorize formulas. Just know the
basics.
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