Download DNA Replication, Recomb, Etc. II

FUNDAMENTALS 1: 10:00-11:00
Scribe: BRANDON TERRY
FRIDAY, SEPTEMBER 10TH, 2010
Proof: THI TRAN
CHESNOKOV
DNA METABOLISM: REPLICATION, RECOMBINATION, AND REPAIR II
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I. DNA Metabolism: Replication, Recombination, and Repair [S1]
II. Outline [S2]
a. The concepts for this lecture you want to know are highlighted in RED on this transcript.
b. They are how RNA genomes and viruses are replicated, how genetic information is shuffled by genetic
recombination, DNA repair systems found in cells, and what the molecular basis is for mutations in DNA.
III. How are RNA Genomes Replicated? [S3]
a. Many viruses have genomes composed of RNA instead of DNA.
b. So, can viral RNA serve as a template for DNA synthesis, and what enzyme could potentially mediate such
processes?
IV. Another Way to Make DNA [S4]
a. In 1964, Howard Temin noticed DNA synthesis inhibitors prevented infections of cells in a culture by RNA
tumor viruses.
b. He then postulated that DNA might be an intermediate in the replication of the tumor viruses.
c. Several years later, Temin’s and David Baltimore’s labs separately discovered RNA-directed DNA
polymerase, aka “reverse transcriptase”.
V. Reverse Transcriptase [S5]
a. All RNA tumor viruses contain a reverse transcriptase.
b. An unusual primer is required to start RNA-dependent DNA replication. It is essentially a tRNA molecule
which the virus captures from the host cells.
c. Reverse transcriptase transcribes the RNA template into a complementary DNA (cDNA) sequence to form a
DNA/RNA hybrid.
VI. Reverse Transcriptase Activities [S6]
a. This slide summarizes three known enzyme activities for reverse transcriptase.
b. These are RNA-directed DNA polymerase (he says RNA-directed RNA polymerase, but I’m sure this is a slip
of the tongue), RNase activity which allows it to degrade RNA in the DNA/RNA hybrid, and DNA-directed
DNA polymerase activity which allows it to make a DNA duplex after RNase activity destroys the parental
viral genome.
VII. Figure, The Structures of AZT [S7]
a. Here you see the structure of 3’-azido-2’,3’-dideoxythymidine (AZT).
b. This is one of the few approved drugs for treatment of AIDS.
c. In the cell, AZT is phosphorylated to yield AZTTP (AZT-5-triphosphate). The phosphate groups are added to
the HOCH2 group on the 5 membered ring to make the molecule look like a substrate analog. This allows it to
bind to HIV reverse transcriptase.
d. Whole cell DNA polymerases have a very low affinity for AZTTP. This allows AZT to be specifically used as a
drug for the treatment of AIDS.
e. HIV reverse transcriptase doesn’t incorporate AZTTP into the growing DNA chain due to the presence of its
3’-azido group. It blocks further chain elongation and therefore prevents elongation of the virus.
f. There are several other compounds which are similar to this, but this one was the first discovery.
VIII. How is the Genetic Information Shuffled by Genetic Recombination? [S8]
a. There are several types of genetic recombination found in cells.
b. The most common is homologous recombination which involves similar DNA sequences.
IX. How is the Genetic Information Shuffled by Genetic Recombination? [S9]
a. There is also nonhomologous DNA recombination which occurs when very different nucleotide sequences
recombine. It occurs at a low frequency in the genome.
b. There is also transposition. This allows for the enzymatic insertion of various transposons (mobile segments
of DNA).
c. Nonhomologous recombination and transposition play a significant evolutionary role, and homologous
recombination plays an important role in the maintenance of a healthy genome.
X. Figure, Meselson and Weigle’s Experiment [S10]
a. Homologous recombination involves similar DNA sequences. It is achieved by the process of general
recombination which requires breakage and reunion of parental DNA strands.
b. The proteins involved in the process of general recombination are RecA, RecBCD, RuvA, RuvB, and RuvC.
c. The first indications that the process of homologous recombination is actually taking place came from early
experiments of Meselson and Weigle where they demonstrated both in vivo and in vitro exchange of
chromosome parts between genomes of different phages (refers back to slide 10). They infected bacteria with
two populations of phages. One was density labeled so it contained a “heavy” genome and another wasn’t
labeled so it contained a “light” genome.
FUNDAMENTALS 1: 10:00-11:00
Scribe: BRANDON TERRY
FRIDAY, SEPTEMBER 10TH, 2010
Proof: THI TRAN
CHESNOKOV
DNA METABOLISM: REPLICATION, RECOMBINATION, AND REPAIR II
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d. Both of these populations infected the bacteria and the progeny was isolated and separated by a density
gradient.
e. Heavy phages precipitated towards the bottom of the tube and light phages floated at the top, but in between
those two populations additional diffuse recombinant phage particles could be found which contained DNA
derived from both light and heavy phages’ genomes. This was the first indication that recombination was
taking place.
XI. Mechanism of Recombination [S11]
a. If you look at the mechanism of general recombination, you see that essentially any pair of homologous DNA
segments can be used as a substrate.
b. In 1964, Robin Holliday proposed a model which involved single-stranded nicks at homologous sites of
recombining DNAs followed by duplex unwinding, strand invasion, and ligation. These processes create a
Holliday junction.
XII. Figure, The Holliday Model for Homologous Recombination [S12]
a. This slide shows a model for Holliday homologous recombination.
b. It starts with two homologous DNA duplexes which are aligned and create a synapsis.
c. Recombination begins with the introduction of single-stranded nicks at homologous sites on two
chromosomes.
d. At the next stage, strand invasion occurs through the partial unwinding and base-pairing between the two
parental strands.
e. Next, free ends are ligated and result in a cross-stranded intermediate which is called a Holliday junction.
f. These branches can migrate along strands of DNA by unwinding and rewinding duplexes.
XIII. Figure, The Holliday Model for Homologous Recombination [S13]
a. Branch migration results in strand exchange.
b. During this process, another pair of nicks has to be introduced in a vertical or horizontal plane resulting in
“patch” or “splice” recombinant heteroduplexes.
XIV.
Enzymology of Recombination [S14]
a. The process starts with a protein called RecBCD which initiates recombination in bacterial cells.
b. The RecA protein forms a nucleoprotein filament which allows for strand invasion and homologous pairing.
c. The Ruv proteins drive branch migration and help to resolve the Holliday junction into recombination
products.
d. Most of the facts known about homologous DNA recombination are derived from studies in bacteria, but
similar homologous proteins are found in eukaryotes. It is assumed that eukaryotic systems are probably
similar.
XV. Figure, Model of RecBCD-Dependent Initiation of Recombination [S15]
a. The RecBCD enzyme consists of three subunits which have helicase and nuclease activities.
b. It also has a chi site which is a recombinational hotspot. More than 1000 of these are found in the genome of
E. coli.
XVI.
Figure, Model of RecBCD-Dependent Initiation of Recombination [S16]
a. RecBCD binds to a duplex DNA end and its helicase activity starts to unwind to duplex DNA. This creates
“rabbit ears” of single stranded DNA.
XVII. Figure, Model of RecBCD-Dependent Initiation of Recombination [S17]
a. This single stranded DNA is bound by single stranded DNA binding protein and occasionally RecA protein.
XVIII. Figure, Model of RecBCD-Dependent Initiation of Recombination [S18]
a. At the next stage, RecBCD moves along the DNA until it encounters a chi site.
b. After it encounters a chi site, it cleaves the 3’ terminal strand just below the 3’ end of chi.
XIX.
Figure, Model of RecBCD-Dependent Initiation of Recombination [S19]
a. At this point, RecBCD drives the binding of the RecA protein to the 3’ end of the DNA. This binding of RecA
allows strand invasion of a homologous DNA strand.
XX. The RecA Protein – Recombinase [S20]
a. It is a rather small 38 kD enzyme that catalyzes ATP-dependent DNA strand exchange.
b. It forms filaments with deep grooves that can accommodate DNA strands.
c. The complex of RecA with single stranded DNA can also bind double stranded DNA at the secondary site
and search for regions that are homologous with the single stranded DNA bound with RecA and form a
homologous DNA duplex.
XXI.
Figure, The Structure of RecA [S21]
a. This slide shows the ribbon structure of the RecA protein. On panel b, you can see the construction of the
RecA filament.
b. Each RecA filament has four helical turns. Each turn consists of six molecules of RecA protein.
FUNDAMENTALS 1: 10:00-11:00
Scribe: BRANDON TERRY
FRIDAY, SEPTEMBER 10TH, 2010
Proof: THI TRAN
CHESNOKOV
DNA METABOLISM: REPLICATION, RECOMBINATION, AND REPAIR II
Page 3 of 5
XXII. Figure, Model for Homologous Recombination [S22]
a. RecA protein and SSB help with strand invasion of the 3’ single stranded DNA into a homologous DNA
duplex forming a D-loop.
b. The D-loop strand is later displaced by strand invasion and pairs with a complementary strand in the original
duplex to form a Holliday junction as strand invasion continues.
XXIII. Resolving Holliday Junctions [S23]
a. Three proteins are important for this process.
b. RuvA and RuvB both act as a helicase which help to dissociate the RecA filament and catalyze branch
migration.
c. RuvC is an endonuclease that binds to the junction very tightly and cuts pairs of DNA strands of similar
polarity.
XXIV. Figure, Model for Resolving Holliday Junction [S24]
a. The RuvA tetramer fits tightly with the junction point.
b. RuvB hexameric rings have helicase activity and they assemble on opposite sides of DNA heteroduplexes
and act as motors to promote branch migration.
c. RuvC aka resolvase binds to the Holliday junction and cuts it by its nuclease activity.
d. These patch or slice recombination products can be produced dependent in which direction resolvase cuts
the DNA strands.
XXV. Figure, Knockout Mice [S25]
a. This figure shows a practical use for how homologous recombination can be used in the lab. This is done to
investigate the essentiality of a gene based on homologous recombination by creating knockout mice.
b. If you have a gene, you can disrupt the exons by incorporation of a new gene. This disrupts the gene’s
activity.
XXVI. Figure, Transgenic Animals [S26]
a. The resulting construct is inserted into embryonic ES cells. Due to the process of homologous recombination,
the construct can recombine with the parental genome. It can replace the host target gene with the new gene.
b. Due to the presence of this new gene, new clones can be selected and become antibiotic resistant and be
incorporated into mice. You can then test whether this gene is lethal as stages of development are directed
by it.
XXVII. Transposons [S27]
a. They were discovered in 1950 by Barbara McClintock who first showed that activator genes in corn could
move freely about the genome. People didn’t believe her at first, but it was proven 20 years later by a
molecular biologist, and she received a Nobel Prize in 1983.
XXVIII. Figure, The Typical Transposon [S28]
a. The typical transposon has inverted nucleotide repeats at its termini. The transposon acts at a target
sequence by creating a staggered cut, incorporates it into a genome, and then the gaps at the target site are
later filled by polymerase and ligase.
b. Sites of transposon insertion can be recognized by direct repeats in the genome.
c. Transposons have very high evolutionary value.
XXIX. Can DNA Be Repaired? [S29]
a. DNA must be preserved at all costs, as it cannot be replaced like RNA, proteins, or lipids.
b. The human genome has about 150 genes associated with DNA repair.
c. There are multiple DNA repair systems including direct reversal damage repair, single-strand damage repair,
double strand break repair, and translesion DNA synthesis.
XXX. Can DNA Be Repaired? [S30]
a. One of the most common repair systems is the double-strand break repair system.
b. It is of particular interest to genome stability, because lost sequence information cannot be recovered from
the same DNA.
c. Chemical reactions that reverse the damage, returning DNA to its proper state, are direct reversal repair
systems.
d. Single-strand damage repair relies on the intact complementary strand to guide repair.
e. There are also systems repairing single-strand breaks including mismatch repair, base excision repair, and
nucleotide excision repair.
XXXI. Can DNA Be Repaired? [S31]
a. This slide shows double-strand repair through nonhomologous DNA end joining.
b. An important protein for this process is Ku70/80. It binds to the ends of broken DNA and recruits a set of
proteins that juxtaposes the broken ends.
c. These processes tend to generate a proper substrate for DNA dependent protein kinase and DNA ligase IV
which allows ligation and end joining.
FUNDAMENTALS 1: 10:00-11:00
Scribe: BRANDON TERRY
FRIDAY, SEPTEMBER 10TH, 2010
Proof: THI TRAN
CHESNOKOV
DNA METABOLISM: REPLICATION, RECOMBINATION, AND REPAIR II
Page 4 of 5
d. Double-strand breaks that arise during DNA replication can be paired through homologous recombination.
XXXII. Can DNA Be Repaired? [S32]
a. Homologous recombination creates a D-loop due to strand invasion, and sister chromatid-directed DNA
replication restores the information of the damaged duplex.
b. Depending on how the Holliday junctions are resolved by resolvase, you can get either noncrossover or
crossover recombinants.
XXXIII. Can DNA Be Repaired? [S33]
a. A DNA lesion first creates a stalled replication fork.
b. When a lesion occurs at the leading strand, the leading strand cannot continue.
c. Lagging strand DNA synthesis can continue.
d. When Okazaki fragments are ligated, a new strand can invade the new DNA duplex by creating a D-loop and
by strand exchange occurring. In this case the leading strand switches to the lagging strand which uses it as
a template and goes back to the original strand and the stalled replication fork resumes movement.
XXXIV. Mismatch Repair Corrects Errors Introduced During DNA Replication [S34]
a. Mismatch repair allows to correct errors that were introduced during DNA replication. It scans duplexes for
mismatched bases, excises the mispaired region and replaces it.
b. It does this by a methyl-directed pathway in bacteria.
c. Since methylation of DNA occurs post-replication, repair proteins (MutS, MutH, MutL) can identify the
methylated strand as a parent strand and remove mismatched bases on the other strand and replace them.
d. Later after DNA replication and repairs happen, methylation happens again and marks both strands. This is a
rather complicated process.
XXXV. Reversing Chemical Damage (Excision Repair) [S35]
a. This occurs when pyrimidine dimers formed by UV radiation can be repaired by photolyase.
b. It can also occur in excision repair when DNA glycosylase removes damaged base, creating an “AP site”.
XXXVI. Figure [S36]
a. UV irradiation causes dimerization of adjacent thymine bases. This creates cyclobutyl rings between the
carbons of 5 and 6 adjacent pyrimidine rings. Normal base paring in this case would be disrupted. Photolyase
can break cyclobutyl rings.
XXXVII. Figure, Base Excision Repair [S37]
a. When you have a damaged base on one strand, it can be excised from the sugar phosphate backbone by
DNA glycosylase creating an apurinic acid site (AP site).
b. At the next stage, endonuclease severs the DNA strand, and an excision nuclease removes the AP site and
several adjacent base pairs.
c. DNA polymerase I then fills the gaps, and DNA ligase links the chains together completing the repair.
XXXVIII.
What is the Molecular Basis of Mutation? [S38]
a. Multiple point mutations can arise from inappropriate base-pairing.
b. Mutations can be caused by base analogs.
c. Chemical mutagens may react with bases in DNA.
d. Insertions and deletions in DNA can result in frameshift mutations.
XXXIX. Types of Mutations [S39]
a. Types of mutations include large chromosomal deletions, translocations when chromosomal segments are
swapped during mistakes in the process of general recombination, point mutations including transitions and
transversions (a transition is the replacement of a purine with a purine or a pyrimidine with a pyrimidine, a
transversion is the replacement of a purine with a pyrimidine or a pyrimidine with a purine), missense
mutations where one base is replaced with another, and frameshift mutations which is the addition or deletion
of a single base or bases which alter the reading frame of the genome and also alters protein sequencing.
XL. Figure, Examples of Point Mutations [S40]
a. Different point mutations result from base mispairings in unusual circumstances. In panel A, and imino isomer
of adenine can base pair with cytosine instead of thymine. This can result in a change of sequence after DNA
repair is completed.
b. In panel B, adenine in the syn conformation can pair with guanine.
c. In panel C, thymine and cytosine form a base pair by hydrogen bonding interactions mediated by a water
molecule.
XLI.
Figure, 2-Aminopurine [S41]
a. 2-aminopurine is an adenine analog that normally base pairs with thymine but may also base pair with
cytosine through a single hydrogen bond.
XLII.
Figure, Another Example of Base Analogs [S42]
a. Oxidative deamination of adenine in DNA yields hypoxanthine, which base-pairs with cytosine, resulting in an
A-T to G-C transition.
FUNDAMENTALS 1: 10:00-11:00
Scribe: BRANDON TERRY
FRIDAY, SEPTEMBER 10TH, 2010
Proof: THI TRAN
CHESNOKOV
DNA METABOLISM: REPLICATION, RECOMBINATION, AND REPAIR II
Page 5 of 5
XLIII. Figure, Examples of Chemical Mutagens [S43]
a. There are many chemical mutagens that can affect many different bases resulting in various kinds of
mutations.
b. Many of the mutagens listed on the slide are commonly used in labs to introduce mutations into model
organisms.
XLIV. Example of a Frameshift [S44]
a. A frameshift occurs when addition of a single base completely changes the sequence of amino acids,
resulting in an inactive protein.
XLV. Diseases of DNA Repair [S45]
a. There are many diseases such as those on this slide that disrupt the process of DNA repair.
XLVI. Review [S46]
a. Know the structure and functions of different nucleotides, important discoveries which helped solve
the DNA structure (ex: Chargaff’s Rule, X-Ray diffraction data from Rosalind Franklin), and major
features of the DNA double helix.
XLVII. Review [S47]
a. Know the structure of DNA, how it can be assembled into the A, B, or Z structure, and what the
primary, secondary, and tertiary structures of DNA are.
XLVIII. Review [S48]
a. Know the features of DNA replication (includes the unwinding of the DNA double helix, how it is
semiconservative and semidiscontinuous, and how it is bidirectional), the enzymology of DNA
replication (what enzymes are important for DNA replication to take place), and the difference
between eukaryotic and prokaryotic DNA replication.
XLIX. Review [S49]
a. Know the Enzymology of DNA recombination, the major types of DNA recombination and repair
(Homologous, Non-Homologous, and Transpositions), types of DNA recombination and repair, and
the molecular basis of mutations.
[End 38:31 mins]