BioReg 2004
Due: Tuesday, January 27, 2004. 5pm.
Turn a HARD COPY into Rachel at her desk (GH N374, Madhani Lab)
Hey gang! Welcome to your first problem set. There are 3 main problems – they are mostly based on course material, but also draw from some supplemental papers and some creative thinking  We encourage you to work in groups.
Please be concise when answering these questions. The number of points assigned to each question should give you an idea of how detailed the answer should be. The spacing left between each questions is the suggested length for your answer. You are welcome to type answers into the Word document available online, but remember- be concise. If you handwrite, make it legible. Begin each main problem on a separate piece of paper.
Please don’t staple your entire PS together – keep each main problem separate (for ease of grading) and make sure your name is on each page.
If you have questions, please contact us. Your best bet is to e-mail the person who wrote the question (listed at the beginning of each question).
Liam: Morgan Lab – lholt@itsa.ucsf.edu
Becky: Johnson Lab – rzordan@itsa.ucsf.edu
Rachel: Madhani Lab – rtompa@itsa.ucsf.edu
Good luck!

Part One: Liam’s Question (lholt@itsa.ucsf.edu)
1.1) You have discovered a new organism from the scorched earth around Chernobyl. It is a previously uncharacterized strain of bacteria. You like names. You name it Bacillus radii (BR). You like DNA polymerase. You wish to purify a DNA polymerase activity.
BR iefly describe how you would assay for DNA polymerase activity in crude extracts.
What is your read out? What kind of template would you use? How would this affect your result? [5 points]
1.2) Assuming that your activity is essential for DNA replication in BR, BR iefly describe a genetic strategy that, in conjunction with the assay you have designed, could create mutants for your DNA polymerase. Nobody has offered to pay for a ski trip. No one even offered to buy you a drink. Your in vitro assay is time consuming and fills your radioactive waste bucket in no time. You want to design your strategy to enrich for replication mutants to the greatest possible extent prior to embarking on in vitro work.
[5 points]
1.3) Give one reason why it is sometimes difficult to purify an activity by fractionation of extracts. [1 point]
1.4) You manage to clone two different polymerase enzymes. You don’t pass up the opportunity to name them. You name them PolX and PolY. You wish to characterize them further. Design an experiment to assay fidelity in vitro . You wish to be quantitative and as unbiased as possible. [5 points]
1.5) You are curious to see how accurately BR replicates its genome. You realize that it will be difficult to be quantitative and unbiased so you just decide to determine the relative mutation rates of BR and E. coli. How might you make this comparison?
[4 points]

1.6) You find that BR is 100 fold more susceptible to spontaneous mutations than E. coli .
You also determine that it is resistant to high doses of UV radiation, gamma radiation and DNA modifying agents:
What kind of DNA damage does UV cause? [1 point]
What kind of damage does gamma radiation cause? [1 point]
1.7) Name two mechanisms by which BR could deal with lesions in DNA during replication in order to overcome the damage caused by each of these types of damage.
[2 points]
1.8) You undertake a kinetic analysis of polymerization by PolX and PolY and determine the rate constants illustrated below when incorporating the correct nucleotide or an incorrect nucleotide.
2 E.DNA
N+2
E.DNA
N
+ dNTP a
1
E.DNA
N+1
+ dNTP b
3
E.DNA
N
+ dNMP a
Rate Constant
1
2
3
Rate when dNTPa is Correct
PolX
300
300
0.06
PolY
200
200
0.05
Rate when dNTPa is wrong
PolX
0.2
0.9
5
PolY
0.02
0.008
3
Which polymerase has a lower fidelity? Describe two ways this difference in fidelity could come about. [5 points]
1.9) PolX has a low processivity. Design an assay to determine polymerase processivity.
[5 points]

1.10) In a microarray analysis, PolX is found to be induced upon UV irradiation and exposure to DNA modifying agents. What is a possible function for PolX? Why do its biochemical qualities suit it for this task? [2 points]
These next questions are a bit trickier. You may wish to help each other out.
1.11) You want to see if BR synthesizes DNA in a canonical fashion and decide to look for Okazaki fragments in vivo . How do you know your products are Okazaki fragments and not just fragmented DNA? [5 points]
1.12) You determine that PolY does make Okazaki fragments. You wonder: does the lagging-strand PolY operate processively from fragment to fragment? That is to say, on completion of an Okazaki fragment, does the lagging strand polymerase remain associated with the fork, transfer to the start of the next OF and then continue or does the lagging PolY dissociate on OF completion and a new PolY associate at the beginning of the next OF? Design an assay to test this in vitro . Discuss any assumptions or problems with your assay. [5 points]
1.13) The Okazaki fragments are short by prokaryotic standards (200bp). You are curious as to why the Okazaki fragments are so short: what determines their length?
You decide to test if it is the concentration of primase. You place the primase gene on two different promoters and achieve one very high expressing and one very low expressing strain. You achieve the results below:
Primase Concentration
Endogenous (350nM)
Okazaki Fragment Length
Low (100nM)
Very Low (10nM)
200 bp
260 bp
500 bp

You make the initial conclusion that primase concentration determines Okazaki length.
You now wish to further test your model. You put primase on a continuously titratable promoter and check OF length at a range of primase concentrations. You achieve this result:
600
500
400
300
200
100
0
0 200 400
600
Is this data consistent with primase determining OF length? Why or why not?
[4 points]
End of Part One

Problem 2. DNA Replication & Protein Machines… (50 points)
Becky’s Problems: E-mail any questions to me at rzordan@itsa.ucsf.edu
During a screen of conditional mutants, you come across a strain of E. coli that is defective for replication. Though DNA replication is normal at both RT and 37°C, you find that your mutant of interest is unable to replicate its DNA above 42°C. You perform in vitro assays to determine the extent of DNA synthesis when the presynthesis and synthesis reactions are performed at a variety of temperatures. With your data, you are able to plot the following graph:
2.1 (5 points) Based on the graph shown above, do you think that your mutated gene is normally involved in the presynthesis or synthesis phases of DNA replication? Why?
2.2a (4 points) Luckily, a friendly postdoc in your lab already has purified the various components of the replication fork from a wildtype strain of E. coli . Because the postdoc is so willing to part with the proteins, you become suspicious of how pure they really are.
The postdoc tells you that each protein is 95% pure. What does “95% pure” mean in terms of your purified components? How do you assay for protein purity?

2.2b. (3 points) How would you use these purified components to determine in vitro which replication fork machinery is defective in your mutant?
2.2c (10 points) Design an assay to detect replication in vitro. What are the expected results of this assay if replication does/does not occur?
2.3a (3 points) You’re excited when you find that a wild-type helicase allows you to get replication at 42°C (woohoo!). However, you decide that you need to be able to run
DNA synthesis assays from purified components of your mutant (no more mooching off your friendly postdoc). What technique would you use to purify your mutant helicase?
Please give some examples of purification methods.

2.3b.
(10 points) Throughout your purification, you rely on a helicase assay to determine which fraction contains the desired activity (and presumably your desired protein).
Describe your helicase assay… what is your template? Do any other cofactors need to be added to the reaction, and if so, what? How do you separate and detect the products of the reaction? What would the data look like?
.
2.4a. While celebrating your isolation of the ts helicase, you accidentally leave the replication assay out on your bench, instead of incubating it at 37°C. You find that replication doesn’t occur at 25°C. You then remember that the priming step of DNA replication is cold sensitive, and doesn’t function at room temperature. Just to be thorough, you decide to run a reciprocal shift assay for DNA synthesis (with your newly purified helicase!), using all combinations of 25°C, 37°C, and 42°C for the presynthesis incubation and the synthesis steps of elongation. Your template for the DNA synthesis assay is a dsDNA circle, which contains and ORI. Assume all proteins required for DNA synthesis (including your ts mutant helicase) are used in every reaction condition
Fill out the chart below. For each row, the reaction is incubated at the temperature indicated in column 1 for a sufficient amount of time, then dNTPs are added and the temperature is shifted to the temperature indicated in column 2 for the duration of the experiment. For each situation, please indicate to what degree DNA replication occurs -- completely, not at all, or partially (please state the intermediate that results, or the DNA product of the partial reaction). Also note the reaction intermediate that could be seen using EM.

+ dNTPS
Col 1 Col 2 Extent of DNA replication
25 25
25
25
37
42
EM intermediate
37
37
37
42
42
25
37
42
25
37
42 42
2.4b. (1 point) Besides the proteins that comprise the replication machinery, what cofactors do you need to include in the beginning reaction to allow the presynthesis process to occur?
2.5. (5 points) Because you have invested so much time in with your mutant strain, you decide to look for suppressor mutations. You further mutate your ts mutant, and you look for isolates that are now able to undergo DNA replication at 42°C. Given your original mutation was in a helicase, give 3 possible means of suppressing this mutation.
END OF PROBLEM 2
#3 – Rachel’s problem (rtompa@itsa.ucsf.edu)

Note – Most of these problems are meant to test your understanding of the class material.
The last part of each problem (**) is trickier; these problems are meant to stretch your brain a little.
3.1) 30 points Chromatin is a many-splendored thing
You are interested in characterizing the establishment of silencing at the silent mating type locus HMR in S. cerevisiae . You know that these loci require both cis and transacting factors in order to be silenced. You decide to investigate the neighboring flanks of
HMR for possible sequences that could confer silencing.
3.1a) 4 pts You decide to look for origins of replication in these regions. Describe how you would map these origins biochemically/physically and genetically (give one example technique of each), and name some drawbacks to each technique.
Biochemical/physical mapping:
Genetic mapping:
3.1b) 3 pts You find sequences that look like origins of replication near the silencers of the mating type loci (see above illustration). You decide to further characterize the timing and efficiency of these loci. You decide to look at these origins by 2D gel after synchronizing your cells in alpha factor and then releasing them. You obtain the following data using specific probes for the two origins near HMR as well as for an origin in a different location:

Characterize these origins as early/late, efficient/inefficient.
A-
B-
C-
3.1 c) 4 pts You find that one of the origins near HMR replicates late in S phase and inefficiently. How would you test whether their chromosomal context affects their replication timing and efficiency (see above diagram of HMR)?
3.1 d) 6 pts You wonder whether progression through the cell cycle is important for the establishment of silencing at HMR, and if so, which stage of the cell cycle may be important. Diagram an experimental outline to test this hypothesis, using a sir3 ts strain
(your experimental read-out is mRNA levels expressed from a gene within HMR,
HMRA1). You don’t have to provide as many details as a lab protocol would, but provide enough details so that a proficient post-doc would be able to perform the experiment based on your description.

3.1 e) 6 pts Show the expected results from part d if a cell cycle event (you pick the phase) is necessary for establishment of silencing (i.e., what do the levels of
HMRA1 mRNA transcript look like at different points in the cell cycle? The simplest way to represent this would be a Northern):
If no cell cycle event is necessary for establishment of silencing:
3.1 f) 7 pts You find that passage through S phase is required for establishment of silencing. You decide to test whether DNA replication may be the event needed for establishment of silencing. i) How would you test whether firing of the origins flanking HMR is necessary to establish silencing?

** ii) How would you test whether a replication fork actually needs to pass through HMR in order to establish silencing?
3.2 20 points Phun with phosphorylation
3.2 a) 6 pts You are studying the role of an essential budding yeast protein, Bioreg1
(Brg1), in DNA synthesis. You isolate a temperature-sensitive mutation of this gene, where cells at the restrictive temperature arrest with large buds and a 1C DNA content.
Thus you suspect that Brg1 plays a role in DNA replication, but you are not sure whether it is important in initiation or elongation. How would you distinguish between the two possibilities?
3.2 b) i. 6 pts You notice that Brg1 contains consensus sites for phosphorylation by Cdk1. You find that Cdk1 phosphorylates Brg1 in vitro.
Showing that Brg1 is a Cdk1 substrate in vivo is very difficult, but what data could you get that would strongly suggest that Cdk1 phosphorylates Brg1 in vivo ?
** ii. 8 pts You found that Brg1 plays a role in replication initiation, and you want to test whether phosphorylation of Brg1 by Cdk1 is important for this role. You make a mutant strain of Brg1 where its Cdk1 phosphorylation sites are mutated, but this strain is dead. You’ve found out that the Cdk1 phosphorylation sites are important for viability, but how would you show that they are important for initiation?