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BIL 255 EXAM 2 QUESTIONS AND VERIFIED ANSWERS
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BIL 255 EXAM 2 QUESTIONS AND VERIFIED ANSWERS.....
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Semiconservative replication - ANSWER Method of DNA replication inwhich parental strands separate, act as templates and produce molecules of
DNA with one parental DNA strand and one new DNA strand
Three Postulated Models of DNA Replication - ANSWER 1. *Semi-
conservative*
2. *Conservative*-one double helix of new DNA and another helix of template
DNA
3. *Dispersive*- there will be breaks between the DNA strand and be a mix of
old and new DNA
Meselson-Stahl Experiment - ANSWER •Invented a new technique to separate
DNA by mass
•Used isotope of nitrogen to change the weight of DNA N15 & N14,
demonstrated that the semi-conservative model is the best description of
replication.
•Bacteria is first grown in a heavy medium, so the parent strand is heavy, then
transfer bacteria to a light-medium where all new DNA strands are lighter
•In semiconservative, the first replication was lighter than the parent strand, and
in the second doubling, there were even lighter strands
Pulse-chase protocol - ANSWER •Do a density centrifuge experiment by
adding DNA to a salt (cesium chloride) solution which makes a gradient and
separates thing of similar mass
•The N15 is more dense and closer to the bottom of the tube compared to N14
What would the pattern of N14:N15 DNA have looked like after *one
generation* if the replication pattern was dispersive? - ANSWER a) There
would have been one band
What would the pattern of N14:N15 DNA have looked like after two
generations if the replication pattern was dispersive? - ANSWER a) There
,would have been 1 band (or a smear) since there isn't equal dispersion between
each strands after the second replication
How is DNA double helix opened up? - ANSWER •Initiator proteins bind to
specific DNA sequences called *replication origins*
-Separate a short length of DNA a few base pairs at a time therefore does not
require a large energy input since each h-bond is weak individually
-Usually *start in A-T rich stretches of DNA* since they have fewer h-bonds
holding them together
•When the DNA synthesis occurs, the replication origins are further opened up
How are new nucleotides incorporating in a growing chain? - ANSWER •The
nucleotide first goes to its complementary base
•A phosphodiester bond between the 3ʹ end of the growing DNA chain and the
5ʹ-phosphate group of the incoming nucleotide, which enters the reaction as a
deoxyribonucleoside *triphosphate* (hydrolysis of high-energy phosphate bond
is used to power the polymerization and releases pyrophosphate (2 phosphates)
which is then further hydrolyzed to *inorganic phosphate* (Pi) which is
irreversible)
•The *chain grows in the 5' ➝ 3' direction* so it is complementary to 3' ➝ 5' of
the template
What different proteins does DNA synthesis require? - ANSWER • *DNA
template* (parental DNA)
• *dNTP's* (dATP, dCTP, dTTP, dGTP) are the different nucleotides with the
triphosphate
• *Enzymes*
- Helicase
- Topoisomerase
- Single Strand DNA Binding protein (SSB)
- Clamp
- DNA dependent DNA polymerase (DNA polymerase)
- Primase (RNA polymerase)
• *Primer*! (Are a short sequences of already assembled RNA nucleotides;
RNA polymerases do not need it)
,DNA dependent DNA polymerase - ANSWER •*Enzyme that catalyzes the
formation of the new DNA strand* with the attachment of the 5' of the incoming
nucleotide to the 3' end of the previous one with a phosphodiester bond
•*Many sub-types *
-Some are used for replication while others are for repair
• Highly processive (up to 1000 bp/sec)
•Two catalytic sites:
- Polymerizing or P site
- Editing or E site
• Synthesis 5'-3' direction (3' -> 5' of old strand is complement)
What direction does the replication fork go? - ANSWER Bidirectional
DNA polymerase P (active) site - ANSWER • The complement nucleotide
comes in and the polymerase and is first matched up via hydrogen bonds, then a
phosphodiester bond of the backbone is formed by the polymerase
• *Polymerizing site*
• Error rate of 1 error every 1x10⁵ bases.
• "Proofreading" mechanism:
- Base pairing leads to *conformational change*
- The conformational change must happen for bond formation to happen
DNA polymerase E (active) site - ANSWER • *Editing* site (exonucleolytic
proofreading)
• 3'-to-5' exonuclease
• Stalling mechanism in P brings misincorporated nucleotide to E site
• It cleaves any incorrect nucleotides and then confirmation returns to normal
• Decreases error rate 100 times to 1 error every 1x10⁷ bases.
Why does the chain grow 5' → 3'? - ANSWER If extension proceeded in the
opposite direction (3' → 5'), proofreading mechanisms would terminate chain
elongation
Leading strand has ___________ synthesis. - ANSWER continuous
Lagging strand has ___________ synthesis and is composed of _____________
__________. - ANSWER discontinuous; Okazaki fragment
, Primase (RNA Polymerase) - ANSWER • An enzyme that joins RNA
nucleotides to make the primer 10 nt long using the parental DNA strand as a
template without the need of a hydroxyl group, so then the DNA polymerase
can take over
• Has to make a primer for every Okazaki fragment
RNase - ANSWER Degrades RNA primers
DNA Ligase - ANSWER An enzyme that eventually joins the sugar-phosphate
backbones of the Okazaki fragments
Why is RNA synthesis used to make a primer? - ANSWER c) RNA doesn't
require a free 3' hydroxyl for synthesis
DNA Helicase - ANSWER • It's a protein machine that moves bidirectional
and has six proteins that move in a concerted fashion via hydrolysis which
changes it's conformation
• It is composed of six identical proteins and is asymmetrical, where two of the
bindings sites bind to ATP, another two ADP + Pi, and the last two are empty
• *Opens dsDNA AFTER the initiator proteins start the break*
• Requires ATP
• 5'-to-3' and 3'-to-5' helicase
Topoisomerases - ANSWER • Due to torsional stress caused by the DNA
helicase, super coils forms which is bad since it makes the DNA less accessible
for replication
• The topoisomerase make DNA nicks a bit upstream of the parental DNA helix
to prevent supercoils from forming and a ligase comes and closes the breaks
Single stranded DNA binding protein (SSB protein) - ANSWER • Binds to
single stranded DNA
• Prevents hairpin loop formation
• Exposes DNA
•*Keeps the side chains from reacting with other far off side chains
•More important in the lagging strand
Clamp protein - ANSWER • Clamp allows DNA polymerase to remain
attached to DNA for long stretches, so it's more important for the leading strand
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