BMCB625:pol-Y (Excision Repair): Difference between revisions
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===Chayne=== | ===Chayne=== | ||
In the discussion, the authors draw upon kinetic data to support their conclusions that pol k can catalyze repair synthesis 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 (~100 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. | |||
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===Jon=== | ===Jon=== | ||
Revision as of 16:27, 13 June 2007
(Homework) Questions
Mahta
Chayne
In the discussion, the authors draw upon kinetic data to support their conclusions that pol k can catalyze repair synthesis 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 (~100 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
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?
