Download Answers to Problem Set 3A

Problem Set 3A Answers Name and Lab Section:_____________________
Due 10-16-06 at beginning of lecture
1. (Read the first 3 questions before you do any of them.) Draw a section of the E. coli
chromosome as replication is occurring through that region. Give yourself plenty of
room and make the replication bubble stretch most of the way from one side of your
paper to the other. Show both replication forks and a bit of DNA at each end that has not
yet been replicated. Use single long arrows for each of the original DNA strands, being
sure with all DNA and RNA strands that the arrows are oriented correctly to signify the
5’ and 3’ ends of the molecules. Identify the origin of replication with “ori.” Use long
arrows to signify the leading strands; wavy arrows (or wavy parts of lines) to signify
RNA primers; and short arrows to signify lagging strand DNA.
2. List (and number) 8 enzymes known to be involved in DNA replication in E. coli and
give a single phrase, or sentence description that distinguishes what each of them does.
The description should have enough detail to distinguish each enzyme from any of the
others involved in DNA synthesis.
1. Initiator protein: Binds to origin and separates strands of DNA to initiate
replication.
2. DNA gyrase (topoisomerase): reduces supercoiling of the DNA ahead
of replication fork by making, then resealing breaks in the deoxyribosephosphate DNA backbone.
3. Helicase: Denatures DNA at replication fork.
4. Single-strand binding proteins : keep DNA single stranded.
5. Primase: an RNA polymerase that uses DNA as a template to
make RNA primer for DNA replication.
6. DNA polymerase III: elongates primers in the 5'--->3' direction, giving
continuous synthesis on the leading strand and discontinuous synthesis on
the
lagging strand.
7. DNA polymerase I: digests RNA primers with its 5'--->3'
exonuclease activity and replaces with DNA.
8. DNA ligase: joins unlinked DNA strands by sealing nicks in the sugar
phosphate backbone.
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Note that the enzymes (numbers) are placed in locations they are likely to be
functioning with respect to the way the DNA molecules are presented (ie, the
topology of the DNA). This is a simplified view. For example, you could have
placed DNA polymerase III (the circled number 6) at either of the two locations
marked in the figure, because with respect to the DNA as its drawn, it would be at
both of those locations, doing leading strand synthesis at the further right location,
and doing lagging strand synthesis at the left location. Yet we know that both DNA
polymerase III complexes involved in synthesis of the right fork are in actuality at
the same location, and this is allowed because the lagging strand synthesis comes
from a region of DNA that is looped out, then back to the DNA polymerase III
complex. We have drawn it as above to keep the drawing of the DNA simpler. The
same thing would also be occurring at the left replication fork, but in this question
you only needed to show one most likely location of DNA polymerase III.
Notice that DNA polymerase I (number 7) is shown at that particular
location because the RNA primer is shorter there, signifying that it has already
digested away part of the RNA primer as it’s synthesizing new DNA to replace the
RNA. Similarly, the primase is at number 5 because that RNA primer is not yet
full-length, signifying that the primase is still synthesizing the rest of that primer.
DNA ligase is at number 8 because there is no RNA primer left there, yet the
two new strands have not yet had their phosphate/deoxyribose backbones sealed, ie,
there appears to be a nick there.
At the point of replication in this figure, the initiator protein would not be
likely to be functioning anywhere, because it would have finished its job. Yet if it
were to still be bound anywhere, the most likely location would be the origin. One
reason for this is that if replication is occurring extremely rapidly, the initiator
protein might already be bound at the ori (origin of replication) getting ready to
start the next round of replication.
The listed locations of the helicase and DNA gyrase are probably need no
explanation.
3. In the figure you have drawn for question 1, place a number encoding a most likely
location where each of the above enzymes would be functioning at the time of the
drawing.
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4. What does telomerase use as template for synthesizing more DNA on the ends of chromosomes?
What does it use as primer?
It uses its own RNA as template and uses the 3’ hydroxyl on the DNA at the end of the
chromosome as its primer.
5. Why do Ds elements need the presence of an Ac element in order to transpose?
Because the transposase gene in Ds elements is at least partially deleted and thus
nonfunctional, so they need to use a transposase protein produced by an Ac element.
6. What is the function and structure of most centromeres?
They serve as attachment sites for the spindle apparatus for the point of movement of
chromsomes during cell division, allowing proper chromosomal segregation. Most
centromeres are composed of many copies of short tandem repeats.
7. In the process of sequencing a portion of an organism’s genome, you identify a number of
regions of about 6 kb (kilobases), each of which has direct repeats at the ends of it. The direct
repeats are unique for each of these regions. For each element, just inside of the direct repeat
at one end is a region with 40-50 A’s in a row (polyA repeat). You find a relatively long ORF
(open reading frame) in the 6 kb region. What does that ORF probably encode? What does
this tell you about the nature of the nucleic acids which moved each of these elements to their
present location?
The ORF probably encodes a reverse transcriptase. This means that this element is
probably a retrotransposon and moved via production of an RNA intermediate, then
reverse transcription of that RNA into a DNA copy.
8. List four types of middle repetitive DNA in eukaryotic genomes.
Here are five types:
1. SINEs, which are Short Interspersed Elements (an Alu is one example)
2. LINEs, which are Long Interspersed Elements (a LINE-1 is one example)
3. Retrovirus-like elements (these normally still have their LTR’s)
4. multicopy genes (histone genes and ribosomal RNA genes are two examples)
5. gene families, each family consisting of similar but not identical genes (the
globin genes and immunoglobulin genes are examples)
9. What type of exonuclease activity do DNA polymerases that can do proofreading have?
Why does that activity increase the accuracy of DNA replication?
A 3’Æ5’ exonuclease activity. This activity allows them to digest away an incorrect
nucleotide that has just been placed into the 3’ end of a growing DNA strand. This gives
the extension activity of the polymerase a second chance to insert a correct nucleotide.
10. What are the four things that enzymes involved in recombination probably need to be able
to do?
1. nick DNA
2. help complementary strands base pair in new combinations
3. small amounts of DNA strand synthesis
4. resolve intermediate structures formed by doing further nicking and ligation
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