Week 2 - High speed of fork progression induces DNA replication stress and genomic instability
Layman abstract: DNA replication is a complex, finely-tuned process that is regulated by a wide range of proteins. One such group of proteins are poly(ADP) ribose polymerases, also known as PARP proteins. PARP proteins have been shown to be an important part of recognizing and repairing DNA damage that occurs during replication. Inhibition of these proteins has proven to be an effective therapy in certain cancers, including subtypes of breast and ovarian cancers. In this paper, the authors show that the therapeutic effectiveness of PARP inhibition might be due to how nullifying these proteins causes an acceleration in DNA replication. At high enough speeds, some DNA repair pathways cease to function properly. This dysfunction is overcome by backup repair pathways in healthy cells but leads to cell death in tumors that lack those same repair pathways. These results highlight the relevance of replication speed in the scope of DNA replication damage control and the authors propose how PARP proteins may have some regulatory control over such speed.
- The authors mention that their findings apply to both cancer and aging. They make the case for cancer, but how do their findings relate to aging?
*Breanna: I would assume it has to do with cancer and aging are very linked through the use of DNA damage repair pathways. If PARP inhibition causes BRCA (part of dna damage repair pathway) deficient cells to fail, it would also make sense that it could work similarly in aging. I also read outside of this paper that PARP is part of the BER pathway (specifically detection of breaks and signaling for repair) which again is important in both ageing and cancer *I'm not an expert tho so I could be totally off*
- All of the experiments were done in a cell line would this data be supported in primary cells?
- TUNEL positivity in cells is usually used as an indicator for apoptosis. Do these cells undergo apoptosis after PARPi?
- They briefly mentioned that PARP1 physically binds the P21 protein, but what does it do? They mention that PARPi inhibits nuclear localization of P21, so does it PARylate P21 to localize it to the nucleus?
- What is the mechanism for PARP regulation of fork speed progression?
- Did the authors discuss what breaks down when the fork speed becomes too fast? If not, what makes determines the threshold for "too fast"
- Is helicase or polymerase the rate limiting step for replication?
- Does temperature affect the rate of replication?
- Do slower polymerases generally have higher fidelity?
- The authors are studying PARP and fork progression, is it safe to assume that all cells they obtained data from are in S phase? I didn’t see any cell cycle analysis or control.
- How did they measure fork speed and symmetry?
- Is there any disease related to mutations in PARP?
- Can DNA pol moderate the speed it moves along the DNA?
- Possible answer (by Kathrina): It probably can; DNA pol has exonucleolytic activity and it could perhaps "feel" if incorrect bases have been added.
- What factors change DNA pol's speed? Is the replication fork speed limited by DNA helicase, etc.? What is the limiting factor?
- What role do the other PARPs play? Could non-catalytic PARPs be involved?
- At times it was very confusing to follow the experiments and inhibition pathways they used. I feel like maybe the authors could have put in similar figures to Figure 4k to help visualize how they are modifying the PARP pathway.
- I'm a bit confused about one of the methods. How does the TUNEL assay work and what are they looking for by using this method?
- Julia: TUNEL assays screen for breaks in DNA. I believe the assay works by adding fluorescently labeled dUTP and the enzyme terminal deoxynucleotidyl transferase (TDT) which catalyzes the addition of the nucleotide to any free 3'-OH. Since the dUTP is then added to any free 3'-OH groups, it is effectively labeling any fragemented DNA which would have free 3'-OH groups. It is usually used to detect apoptotic cells (I think Haley touched on this a bit in her comment above) so I'm also wondering about Haley's question regarding the fate of the PARPi treated cells...
- Why did the authors not really look at other cell cycle stages in their analysis other than the S phase?
- The authors do mention that in one of their experiments, they looked at the PARP1 knockdown affecting fork rate and looked at the "cell cycle towards G1 enrichment." Why would they be looking at the G1 phase instead of the S phase in this case?
- They mentioned that PARP1 inhibitors would enhance deleterious effects? Do they mention the deleterious effects like increased cell death, mutation, and increasing the progression of cancer and aging of cells?
- How come fork acceleration is independent of cell type?
- Possible answer: all cell types, regardless of what type, have the same replication and DNA damage repair response proteins/pathways.
- Have there been protein-protein interaction assays to check to see which PARP proteins may be redundant to each other?
- What does PARP2 do in the elongation process? Is it a redundant protein of PARP1? And isn't it a given that a disabled p53-p21 interaction promote fast elongation if those two proteins inhibit DNA synthesis? (Referring to line: "We predicted that in cells that lack PARP2 and with disabled p53–p21, forks should elongate fast even after PARP1 knockdown, because PARylation might not be maintained and p21 may remain low.")
- How measure the distance between origins of replication?
- why use U2-OS as sample?
- At the end of the paper it conclusion present concept is also relevant for ageing? How it would be for ageing?
- Cancer cells are more difficult to treat because they often leave room for adaptation, especially under a selective pressure like the PARP inhibitors. I wonder if long term, adaptation studies would show cancer cells activating other repair mechanisms to decrease the effect of these PARPS.
- They also mentioned the use of PARPS in conjunction with chemotherapy. I wonder if there is a transcriptional mechanism that a small molecule can perturb to add another instance of mutagenesis within the cancer cells.
- How exactly does the PARPS not effect normal cells as much?
- Taking these findings into account, could olaparib or other PARP inhibitors find justifiable use against other forms of cancer, other than BRCA-dysfunction derived cancers?
- Do we see analogous fork-speed regulation mechanisms such as PARP in prokaryotes? How does their average replication fork speed compare to eukaryotic cells?
- Are there any observed cases of fork-speed that is too low? Would that phenomenon similarly trigger DDR pathways, or simply result in slower-replicating cells?
- If PARPs are important parts of DNA damage repair pathways, wouldn't employing PARP inhibitors increase the risk of an offsite secondary cancer?
Answer from discussion: Yes, but chemotherapy also has this issue so our tolerance for that risk is high.
- Can someone explain a Comet Assay?
- Considering PARPs are part of an important DNA repair pathway, have the PARP genes been investigated as potential oncogenes? Are there diseases known to be a result of a PARP mutation? If you can inhibit them without catastrophic results, I would think a mutation wouldn't outright be embryonic lethal.
- Is replication speed ever increased to induce mutagenesis for a process like V(d)j recombination?
- Is it possible that PARPs do not lower the general speed at which the fork moves, but instead act as a breaking mechanism which slows/stops the fork when damage is detected?
- Possible answer: I think this would be possible, and the results seen would be similar. I don't know a lot about DNA repair and the methods discussed in the paper, so I could just be missing something simple.
- What is being PARylated in the replisome?
- Does PARPi literally speed up elongation or is it simply removing the breaks?
- In what ways does their new model vary from the currently accepted model? And were there reproductions of the current model or is that model just based on a single groups work?
- PARPi inhibits the SSB repair pathway so it makes sense that this would result in cell death in cells with non-functional DSB repair, but does this lead to mutagenesis in healthy cells? Does the DSB repair pathway in healthy cells completely compensate for defective SSB repair?
- How did the researchers here draw the conclusions they did from the comet and TUNNEL assays – how did this help them determine that fork speed was accelerated rather than stalled? Neutral vs basic comet assays – what information do the different pHs give about the DNA damage present?
- What is treslin?
- ssDNA breaks activate PARP1 - how?
- When they talk about “analysing symmetry between first and second pulse in double-labelled DNA fibres”, are the referring to a pulse-chase experiment? How does this work?
- Would an overactive PARP result in increased DNA damage and therefore increased risk of cancer?
- The paper touches on the fact that PARP1 inhibition speeds up replication without stalling the replication fork and comes back to this point a few times. Is there a known limit to replication speed? Would these ~60% speed increases typically result in a stalled replication fork? In other words, is this something to pay attention to, or are speed increases of this magnitude well tolerated at the replication fork (aside from introducing DNA damage.)
- The PARP family consists of 17 different enzymes. Was the PARP inhibitor used in this study specific for PARP1? If not, or if this is unknown, is it possible that there are multiple functions/pathways being affected by PARP inhibitor? How would this affect the interpretation of the results?
Week 1 - The Neuronal Gene Arc Encodes a Repurposed Retrotransposon Gag Protein that Mediates Intercellular RNA Transfer
Layman Abstract: There is little known about the molecular function of Arc or how it was evolved. Currently, Arc is shown to be present in neurons and has been implicated as to play a role in neurodevelopmental disorders. This paper shows that Arc is capable of self assembling into a protein capsid or shell, similar to those assembled to viruses. These Arc based protein capsids encapsulate mRNA in one neuron and then export it to neighboring neurons. This article further suggests that, during evolution, the machinery necessary for this function was possibly repurposed from retroviruses or their ancestors.
- Based on the paper, Arc appears to nonspecifically bind RNA. Why is RNA specificity not important (or is it)? Are there ways to still release a specific signal without RNA specificity?
- Although RNA binding is not specific, RNA is encapsulated in a concentration dependent manner. Arc mRNA is specifically trafficked to the dendrites where translation then occurs. This partially rectifies the non-specificity problem as mRNA becomes highly localized and thus more likely to be transferred.
- The Authors note that in Schizophoran's Arc homologs have undergone multiple rounds of duplication vs. tetrapods that only contain a single copy. Does this confer some kind of benefit? How do expression levels compare between the two clusters?
- How might glia-neuron interactions mediate Arc dependent/EV dependent communication?
- Do these extracellular vesicles fuse with other cell types?
- Do other non-neuronal cells secrete these capsids?
- Julia: This Nature review from earlier this year talks about other forms of extracellular vesicles: https://www.nature.com/articles/nrm.2017.125 It seems like there are some intracellular signaling pathways that make use of mechanisms which are similar to the Arc gene function described in this week's paper.
- How does the expression and secretion change during development?
- Why would the body use these protein heavy capsids to transport RNA, do lipid vesicles not offer the same level of protection to the RNA?
- Wouldn't it make more sense for us to have a transport mechanism that wasn't directly introduced by viruses?
- They mention this being a gene linked to Alzheimers, but how could this mechanistically lead into the formation of B-amyloid plaques?
- Are these capsids found in other locations of the body that act similarly?
- Do these capsids specifically form around mRNA? If so, how is this selected for?
- Were these systems acquired from an outside system like a repurposing of viral machinery?
- Does this system use a receptor-mediated endocytosis or a different mechanism?
- Where do arc capsid monomers bind the mRNA?
- How does this binding promote capsid formation between monomers?
- Is there any immune response from the recipient neuron?
- Are ACBARs endocytosed by glia? Could this mediate synaptic plasticity?
- What is the mechanism of RNA binding induced capsid formation?
- Ashley et al. (2018) found that the darc1-mRNA 3' UTR mediates loading into the capsids.
- Are there regional variations in Arc expression?
- Did the fact that Arc transposons seem to have independently originated in fly and tetrapod lineages speak more to the propensity of Ty3/gypsy retrotransposons to integrate and perpetuate themselves or to the evolutionary advantage conferred to organisms having incorporated and repurposed this signaling mechanism?
- How do the capsids disassemble and release the mRNA once inside the target cell?
- What is the specific response in cells receiving Arc capsids carrying Arc mRNA?
- Did the phylogenetic comparison conducted include non-coding regions of the sequence?
- Is there enough structural similarity between Arc and Gag, such that an infection with HIV might induce antibodies against Gag which are cross-reactive to Arc? ?ould this result in an autoimmune-type disease?
- If there have been multiple round of duplication, are there other genes that have a similar function?
- Within one species like mice (since we know many organisms have independently evolved a protein to have the same effect), are there other genes/retrotransposons in neurons that react like Arc and provide similar phenotypes to Arc? Different phenotypes?
- Does the Arc mRNA get sent out to specific neurons, depending on what the organism is learning?
- Is Arc constitutively expressed?
- Is there a mechanism or reason to why prArc capsids show little specificity for particular mRNA?
- How does the capsid molecules actually bind to the mRNA and how does RNA binding facilitate capsid formation? Does the region where RNA binds to important for transport between cells or is it non-specific?
- The authors predicted that they could use genetically engineer Arc and its ability for the capsid to harness RNA, so in what situations would it be beneficial to deliver RNA into cells without an immune activation response? Would this be important for gene manipulation and drug delivery to immunocompromised areas?
- What role do zinc fingers play and how can a cell compensate for a lack of zinc fingers?
- For the knock out mouse, they mention that they use both male and female mice and noticed no differences between sexes. Why might one expect differences between sexes?
- In figure S2, they used 10% SDS-PAGE Gel to analyze their results. How would the resolution of the gel change if you increased or decrease the percentage of SDS?
- Did Arc arise endogenously or from viral infections?
- It sounds like the hypothesis is that arc genes may have existed before defined viruses even existed (retroviruses, at least). There might have been an ancient divergence where some unicellular organisms favored arc-induced transmission as their primary form of propogation, leading to retroviruses, while others simply treated arc as a utility for their more complex replication pathways, leading to other early eukaryotes. -Colin
- Assuming arc has a role in memory development, is it possible/feasible to raise an arc-KO mice to maturity, then compare its learning ability to wt mice? What tests would you use?
- Julia: Yes, in fact this has already been done! The Arc knockouts were unable to form long lasting memories. This is studied using behavioral "learning" tests -- in this case, a Morris water maze which tests how the mouse is able to use spatial cues to navigate a round pool. WT mice were able to develop spatial "strategies" for navigating the maze, Arc KOs weren't. Check out this paper: https://www.sciencedirect.com/science/article/pii/S0896627306006465?via%3Dihub
- Do patients with neurodegenerative diseases have altered levels of arc protein in their neurons? If so, is the alteration global or localized, or is it different across individuals?
- Cont. from above, how would you be able to measure functional arc levels in patients in vivo? Some form of specific protein labeling
- What is the MVB pathway?and what is the end results of it?
- What are the differences between Cry-EM and Negative Stain EM?
- What is the rational behind using HEK293 cells? why not HEK392-FT cells?
- They mentioned that the knockouts were a "gift" from a collaborator, however, I was unable to find their method of knockout.
- How specific are these capsids for certain cell types, and are they specific to the synapse for neurons?
- If this is not a synapse specific transportation, do these capsids enter systemic circulation?
- What is the diffusion distance, are these impacting adjacent neurons or is it farther reaching?