# data[].attributes.body
could you explain the concept of synthetic lethality again and expand a little on the exercise in lecture 5, particularly the experiment as I am a little confused on this..
Is this correct?
If you were to test PARP in BRAC1 deficient cells then could you do a knockout for both PARP and BRAC1 and see if the cells survive or die?

Yes - if they are synthetically lethal cells should not survive. Synthetic lethality means two pathways that work in parallel, if you only loose one cells can still survive but if you loose both either genetically or pharmacologically cells die.

# data[].attributes.body
Sorry this is not in the spec but just for my interest. I was wondering if the CAG repeat expansion as seen in Huntington's was also prone to R-loop formation? Is this why with more CAG repeats, Huntington's disease is more likely/severe due to the increased number of R-loops and therefore increased DNA damage?

Excellent point, very well done. Yes the CAG repeat expansion in HD, which leads to polQ expansion would also increase R-loops and lead to more DNA damage.

Great thank you so much for your reply :)

# data[].attributes.body
Why is there growth and a faint colour on His- and beta-gal plates respectively for XRCC4?

Biology is never black and white, the faint colour and subtle growth is most likely due to some leaky expression of the markers, but the difference between the negative and positive should be apparent.

# data[].attributes.body
The paper "To live or to die: a matter of processing damaged DNA termini in neurons" has been uploaded twice, both for 'DNA end processing article' and 'DNA single break repair and degenerative disease'. Is this correct or were these supposed to be different papers?

This is correct as this review article covers both topics.

# data[].attributes.body
Does MBB6313 have any additional assessment than just the exam? Or is it identical to MBB313?

It will have additional assessment to make up for the extra credit. Please contact Dr Alvey for details.

It has been decided the assessment for MBB 6313 will be as MBB 313 + an additional short essay question to make up for the extra credit.

# data[].attributes.body
Sorry, I got a bit lost at the end of Lecture 2. What substrates would be used for testing for protein activity? And what is the exact purpose of labelling DNA with AMP? Why does increasing the concentration of APTX create a band shift?

No worries at all. The substrates are DNA oligonucleotides with modifications at the 5'-end to mimic AMP covalently linked to DNA. In order to "see" the oligonucleotide substrate on a gel, you have to label the other terminus (i.e. 3'-end') with a radioactive label or a fluorphore for visualisation. The band shift when you increase the concentration of APTX is due to cleaving the AMP from DNA which releases a 'lighter' substrate (DNA WITHOUT the AMP attached) that can travel 'faster' on the gel, hence the shift in bands in a concentration dependent manner. Hope this is now OK?
Great! thankyou so much!

# data[].attributes.body
Please could you add the PDFs of the exercises for sessions 2 and 4. Currently there is just the feedback to download.

Well spotted, these have now been added.

Thank you so much!!

# data[].attributes.body
Sorry i feel this might be a silly question but i dont really understand the role of adding CPT. Does it inhibit TDP1 directly or just misalign the OH so the strand cannot be ligated? If it just misaligns it, does this mean that Top1-Y cannot be cleaved and there's therefore more TDP1-DNA adducts in the cell which then mean the strands cant be repaired and there are more breaks?

This is not a silly question. CPT inhibits the re-ligation of topoisomerase 1 (TOP1), thus you get more TOP1-DNA breaks, which will need TDP1 to be processed by liberating TOP1 from DNA. So, you are correct in that CPT will increase the number of DNA breaks and thus you can test the effect of the repair enzyme, TDP1. Just be careful not to confuse TDP1 (the repair enzyme) with TOP1 = topoisomerase1 (The enzyme the generates the breaks as TOP1-DNA breaks).

thank you so much that makes much more sense, i think i was confused between TDP1 and TOP1, thank you!!!

# data[].attributes.body
Sorry in advance if this is a massive oversimplification. I understand why the double mutant causes a rescue when you explained it on the video, but when you asked us to think about how we would test it (before you said double mutant) I just thought you could add loads of Parp1 to the cell and observe if this had the same effect of depleting the interneurons; am i just being naive and is this a massive simplification?

You are not being naive, this is a great idea and will test the model in a different way. If you over-express PARP1 you would expect to see MORE toxicity and not a rescue.

ok great thank you very much!! :)

# data[].attributes.body
Towards the end of the lecture you talk about how you can add a his tag in the plasmid and purify it through an affinity column. I'm confused on how the protein is pure if it contains a his tag it shouldn't have?

This is a very good point. A his tag is small, often 6 amino acids, which should retain the function of the protein but allows for efficient purification using Ni beads since all bacterial proteins will not bind the Ni beads. Only his tagged proteins will bind the beads, which can be subsequently eluted by imidazole. Thus, the protein prep is pure because it does not contain all the other bacterial proteins, which did not bind the beads because they are not his tagged.

Thank you!

# data[].attributes.body
In lecture 3 you show that mouse interneurons with a knockout mutation for PARP1 can still survive. How can a cell lacking PARP1 survive when it can't detect and therefore repair DNA damage? Surely undetected DNA damage would accumulate more than usual and cause cell death/malignancy? Are there other enzymes in these neurons that rescue its function?  Or have I just got very confused about how PARP1 works?

You are not confused, this is an excellent question. You are absolutely correct in saying 'other enzymes in these neurons that rescue its function'. In absence of PARP1, PARP2 can compensate for SSB detection to promote the repair and then survival of these neurons. Not just this, people found that germline deletion of PARP1 in mice (i.e. not tissue specific or conditional deletion but deletion in the whole organism) is e mbryonic viable, which means PARP2 is able to compensate for PARP1 absence to promote embryonic viability. The double deletion of PARP1 and PARP2, however, is lethal in mice. Hope this answers your question.

Thank you so much! That's so interesting! :)

# data[].attributes.body
Can I just confirm that all of the 'challenges' explained at the end of lecture 1 (packaging, ribose contamination, base modifications) can cause genome breaks. You describe quite a few but only mention 4 in the summary in lecture 2.
thanks :)

Absolutely, all these challenges can cause genome breaks

# data[].attributes.body
Can I please know the time frame for our exams will be 2 hours or 24 hours? Also will they be open book and done online?

Th exams will be open book 24 hours online. Best of luck.
Thank you:)

# data[].attributes.body
Sorry I got a little bit confused at the end of the lecture. I cannot make a link between  XRCC1 and TDP1,ATPX. could you explain what is the relation or between XRCC1 and these enzymes? do mutations in TDP1 and ATPX impact XRCC1?

Good question. The link is that these 3 proteins interact as part of the single-strand break repair complex. The other link is that their deficiency causes a very similar clinical phenotype which is cerebellar ataxia.

Thank you for your explanation. So now that they are components of the same complex, is there a chance for them to have an impact on each other mechanisms or they follow separate routes?

Yes of course, they are part of the same pathway.

# data[].attributes.body
Having seen that high concentrations of TDP2 can slightly rescue TDP1 deficiency (in the experiment where there was half band shift with TTRAP) i was wondering why can't TDP1 slightly rescue TDP2 if it can work the other way round?
I understand they work on different polarities but was wondering why you can't (half) rescue TDP2 deficiency with high concentrations of TDP1 when you can (half) rescue TDP1 deficiency with high concentrations of TDP2.
I fully understand they have different polarities and aren't interchangable, i was just curious.

Excellent point! I have no clear answer to this question except that in yeast people have found that TDP1 can work on 5'-phsphotyrosine substrates albeit with much lower efficiency. This agrees with your speculation, but you got to remember that yeast do NOT have TDP2, so perhaps TDP1 have more 'broader' roles than in human.

Also, from the crystal structure, the active site pocket of TDP1 can slightly accommodate 5'PY whereas the active site pocket of TDP2 is much less restrictive to 5PY.

Hope this helps.

This is really helpful thank you :) really interesting to see the difference between the two

# data[].attributes.body
Could yo make the lecture 6 online content available to watch? I missed a couple things in the LIVE lecture yesterday.

It is already on blackboard, please see the video below on how to access it.

Enjoy!

# data[].attributes.body
Please can you explain the two approaches to study the protein-protein interaction? I think I got Yeast-2 Hybrid but missed the second one. Also, in what circumstances would you use each approach?

The other one is co-immunoprecipitation (co-IP) whereby you immunoprecipitate protein X with an antibody and then look for protein Y using another antibody in the immunoprecipitate.

These are two complementary approaches. Any yeast-2 hybrid result should be confirmed by co-IP. People often do co-IP only if they know what protein to test and of course if there are commercially available antibodies. Hope this helps.

# data[].attributes.body
If I understood correctly, you mentioned in the end of lecture 3 that a mutation in the essesntial protein XRCC1 results in a viable human but a dead mouse, and the explanation to that is that human patients with this mutation produce little XRCC1 that is sufficient for the embryonic development but insufficient for a normal neurological development during the lifetime, right? . However I still don't get why would it result in dead mice, do mice with this mutation not produce XRCC1 at all?

Absolutely correct. What I meant by 'resulting in dead mice' is the GERMLINE DELETION of XRCC1. What you are suggesting is actually a great experiment which has not yet been done (i.e. generating a mouse model that expresses very little XRCC1, which should be viable but suffer from neurological abnormalities).  Hope this clarifies it?

# data[].attributes.body
I am sorry but could you explain what we are seeing in the figure below again please? Did I understand it correctly that it shows the DNA packaged in a micronuclei structure and the nuclear membrane that is encapsulating it? Does the term micronuclei refer to only the lagging DNA or the lagging DNA + the nuclear membrane?
Sorry, it was a bit unclear for me.

You are correct. The figure shows lagging DNA (stained in blue) packaged in a membrane (stained in red) and the whole structure is called micronucleus.

# data[].attributes.headline
Lecture 2 the action of Aprataxin and DNA-AMP adducts

# data[].attributes.subject
Lecture 2 the action of Aprataxin and DNA-AMP adducts

# data[].attributes.body
The ligation reaction of a SSB that initially failed to process the ends and return the correct chemistry to the termini will result in an abortive ligation reaction which will result in the accumulation of DNA-AMP adducts. And those adducts need the action of Aprataxin to cleave of the AMP from the DNA in order to reset the ligation reaction. But Aprataxin only removes the AMP right? it does not process the ends, so what processes the ends here, are we back to the SSB repair steps again where PARP comes along..etc?

# data[].attributes.body
Could you explain what these bar charts mean once again please?

In the absence of Rnaseh2, you get more rNMP in the genome which leads to more micronuclei....Importantly, those micronuclei that are cGAS positive have more DNA damage

The above panel shows this effect in mouse embryonic fibroblasts (MEFs) and lower panel shows the same effect in another cell line called U2OS cells, which is a cancer cell line.

Hope this is now clearer?

# data[].attributes.body
I'm a little lost on the paper Q1 4&5. Could you give me a little direction as to where to look for what chemotherapeutic agent should be avoided? Or where I can look to find this information?

If the defect is in pathway X then you should avoid giving chemotherapeutics that rely on that pathway to repair the DNA damage induced by the chemotherapeutic. Hope this clarifies it.

# data[].attributes.headline
Can we ask our questions from the reading material here as well? There are some things from the papers that were unclear to me

# data[].attributes.subject
Can we ask our questions from the reading material here as well? There are some things from the papers that were unclear to me

# data[].attributes.body
Thanks

Of course, feel free to ask any question here.

# data[].attributes.body
Sorry I'm a little confused on how to interpret the data from S9.6 immunostaining. What exactly do the red and green readouts represent? If we have a green halo, what data does this give us regarding the experiment?

S9.6 positive signal primarily reflects R-loops, the more you have the more R-loops in the cell. Again, any experiment should be confirmed by an orthogonal assay to be sure.

# data[].attributes.body
From the yeast-two hybrid results we can conclude that XRCC4 can also interact with XIP1, so it is also a part of the SSB repair pathway right? Could we make any conclusions about XRCC1 by conducting experiments on XRCC4? What's the relationship between XRCC1 and XRCC2?

I is not clear from the Y2H that they interact and any interaction should be confirmed by n orthogonal assay such as immunoprecipitation. XRCC4 is NOT a component of SSB repair, it is scaffold for non-homologous end joining for repairing double strand breaks.

# data[].attributes.body
Normally ligase attacks AMP via OH. How does APTX chemically attack AMP to covert the adducts to DNA? Also is there any case the lack of aprataxin to be replaced by another mechanism so we don't have AOA1?

1- APTX is an enzyme that catalyse the the hydrolysis between the AMP and DNA, thus clipping off AMP.

2- Well, redundancy occurs in every pathway, there is the possibility that an enzyme called FEN1 cleaves the DNA liberating AMP and a fragment of the DNA but it is not robust in the nervous system.

# data[].attributes.body
How soon after birth will the cerebellum start degenerating in AOA1 and SCAN1? Is there any chance that patients who produce more XRCC1 will develop the diseased phenotype later? If yes is this any other way to predict that genetically/experimentally?

Excellent question - the onset is late after birth, approx teenage, thus it is a progressive disease. Yes in theory if a patient produces more XRCC1 they should develop the disease later or exhibit milder symptoms but this has not yet been reported.

# data[].attributes.body
Since the excessive activation of PARP will lead to NAD depletion, could we design an experiement to measure the NAD istead of the production of polymers? How could we design such as experiment? Will the provided data from such an experiment be specific enough?

Excellent thinking, yes a great idea. There are biochemical assays available that can measure NAD and they could be equally applied to measure PARP hyper activation....BUT NAD is a substrate for other enzymes too, not just PARP so the outcome will be less assertive than directly measured the PAR polymer.

# data[].attributes.body
How can the phosphotyrosine be in both 5' and 3' termini?

This is engineered using chemical modifications of the substrate, to mimic the physiological linkage that takes place on the 3' (TOP1) or the 5[ (TOP2)

# data[].attributes.body
In the feedback for the first question you are mentioning that we should explain the migration of the OH and Y termini. How we could do that? Also is the P terminus not migrated?

The difference in migration reflects the nature of the 3-terminus, phosphate runs faster.

# data[].attributes.body
On the slide 17 on the gel we said that B is TDP1. But since TDP1 is inactive on 5’, why we still can observe a small amount of conversion?

There is very little activity on the 5-, not completely inactive.

# data[].attributes.body
How can we use the repair TDP1 and TDP2 mechanisms as a prognostic marker for cancer/cancer cells?

If they are absent then we should avoid chemotherapeutics that rely on their presence for repair because they will be too toxic.

# data[].attributes.body
When we inhibit PARP1 in our cells and we have less colonies in some plates, is it safe to assume that the native HR system of the cells is defective or is it a normal procedure and we would naturally expect less HR in some cells in comparison to some others?

HR will be less in HR defective cancer cells.

# data[].attributes.body
You mentioned that heterozygous BRCA2 cells are neighbouring cells of BRCA2 -/- ones, is there any particular reason why this is happening?

Loss of heterozygosity is a common form of cancer formation.

# data[].attributes.body
Why are we using RNA FISH with RNA probes and not FISH with DNA ones?

If you are looking at specific transcripts then use RNA probes.

# data[].attributes.body
Has the exam been confirmed as the 22nd of April? What time will it start? There's no information for this anywhere

Exam will be a 24 hour exam released at 12 noon on the 22nd of April.

# data[].attributes.body
Can I please know if TDP1 cause conversion of TOP1-DNA to 3'OH or to 3'P?

3'-P and it needs PBKP to convert the phosphate to hydroxyl

# data[].attributes.body
I noticed the specimen paper says the word count excludes references. Are we expected to use in text citations and do a proper bibliography in the exam answer?

Not required

# data[].attributes.body
Could you please make the recording of the live session available? I was able to access it before under "Live session-recordings", but it's not available now. Thank you!

It should be available, can you try again?

# data[].attributes.body
I am struggling to answer question 3 on question 1 of the specimen.
'For one of your candidate genes in question [2], describe with

illustrations two experiments to test whether the gene you suggest is

defective.'
I thought of maybe isolating patients cells and adding the gene product to see if there is a difference and also, for mre11, we know it binds with rad50 so we could use a yeast-2-hybrid to see if any gene product is present to produce quantifiable product but I didn't know how to expand upon that?

Rescue of phenotypes by adding back the gene is a good approach but you need to spell out the experimental readout, e.g. comet assay, gH2AX, measuring specific breaks using mass spec, etc.. Each of this should be explained as discussed in the lectures.

Thank you!

# data[].attributes.body
The specimen paper describes Section A as an 'essay' question... Are we expected to answer the 5 questions in the format of an essay answer together? Or just as 5 separate shorter answers?
Thank you

5 separate 'long' answers

# data[].attributes.headline
Demonstrating wider reading in data analysis question

# data[].attributes.subject
Demonstrating wider reading in data analysis question

# data[].attributes.body
I am an MSc student, and I know it is important that we demonstrate we have done wider reading in the exam.

I am finding it hard to think how to demonstrate wider reading, and bring points from that reading into the Section A data analysis questions.

Do you have any suggestions for how to do this? Or should we focus on demonstrating wider reading in Section B?