BMCB625:pol-Y (Excision Repair)

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BMCB625 Advanced Topics in Molecular Biology

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(Homework) Questions

Mahta

Discussion Points

DP1. What additional factors bind initial lesions or adducts. How can it be determined if polk associates with them?

DP2. "Hydroxyurea treatment lowers dCTP and dTTP pools while raising dATP and dGTP pools, resulting in precursor pool imbalances that later the fidelity of DNA synthesis that could exacerbate mutagenesis associated with polk NER." (Kunkel et al., Nature Cell Biology June 2006). Why does this occur? How does this affect the results in this paper?



Chayne

In the discussion, the authors draw upon kinetic data to support their conclusions that pol k can catalyze repair synthesis ("fill the gap" in Chris's words) under nucleotide-limited conditions. Why do the differences in kcat/Km between pol delta and pol k support the conclusions, and why might this be "dangerous to extrapolate" in vivo?

-Under saturating dNTP conc, the velocity of the reactions should be similar as the kcat values for these two enzymes are similar (kcat is proportional to Vmax). However, since the Km for pol k is an order of magnitude lower than that of pol delta, pol k has a higher affinity for substrate (at least for dCTP insertion opposite dG) and will thus be faster at lower concentrations than pol delta.

The caveat to this is that these kinetics were measured without PCNA present. PCNA actually causes a large decrease in Km for pol delta (~20 fold), and probably pol k as well. In a physiological context, the precise effect of PCNA (or any other factors) on affinity will determine whether pol k is really favored at limiting dNTP conc.


Jon

Q1. Why does the X axis change from fig 1b to fig 2a? Are these not the same experiment?

How much DNA (ug) is in a MEF? lets assume 6*10^9bp and each bp is 650g/mole

thus 6.5 pg DNA/cell

Figure 2 B highest -HU number for wt is 500 cpm/ug of DNA

so we get 0.00325CPM/cell

times 10^5 is 325CPM/1e5 cells

Why are the axis diffrent

that meens they gave it WAY more UV right???

how come they didn't subtract the background in 2b? that make it 25% like in 1b not 30-40%

I don't get it. I have to work on my talk.




Chris

Discussion point: PCNA is an important cofactor of DNA polymerases that encircles DNA and serves as a critical bridge in recruiting several factors at the replication fork. A recent PCNA Review in Cell has a summary of numerous interacting proteins, including Polymerase kappa. Usually PCNA requires monoubiquitination to interact with thr Y-family polymerases (including Pol k).

One outstanding question in the field is what is the signal that “helps” decide which polymerase will “fill the gap.” If Ogi & Lehmann were to look at the Ubiquitination pattern of PCNA in thee cells, would it be monoubiquitinated, and if so, would Pol k be associated with this? Could a cross-linking study be done to confirm the association? What might be the disadvantage?



Jeremy

In light of Xeroderma pigmentosum, why might it be odd that polymerase kappa is serving this function?
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