TY - JOUR
T1 - DNA polymerase ε leading strand signature mutations result from defects in its proofreading activity
AU - Johnson, Robert
AU - Prakash, Louise
AU - Prakash, Satya
N1 - Funding Information:
This study was supported by National Institutes of Health (NIH) grant R01-GM129689 (to S. P.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2023 The Authors
PY - 2023/7
Y1 - 2023/7
N2 - The evidence that purified pol2-M644G DNA polymerase (Pol)ε exhibits a highly elevated bias for forming T:dTTP mispairs over A:dATP mispairs and that yeast cells harboring this Polε mutation accumulate A > T signature mutations in the leading strand have been used to assign a role for Polε in replicating the leading strand. Here, we determine whether A > T signature mutations result from defects in Polε proofreading activity by analyzing their rate in Polε proofreading defective pol2-4 and pol2-M644G cells. Since purified pol2-4 Polε exhibits no bias for T:dTTP mispair formation, A > T mutations are expected to occur at a much lower rate in pol2-4 than in pol2-M644G cells if Polε replicated the leading strand. Instead, we find that the rate of A > T signature mutations are as highly elevated in pol2-4 cells as in pol2-M644G cells; furthermore, the highly elevated rate of A > T signature mutations is severely curtailed in the absence of PCNA ubiquitination or Polζ in both the pol2-M644G and pol2-4 strains. Altogether, our evidence supports the conclusion that the leading strand A > T signature mutations derive from defects in Polε proofreading activity and not from the role of Polε as a leading strand replicase, and it conforms with the genetic evidence for a major role of Polδ in replication of both the DNA strands.
AB - The evidence that purified pol2-M644G DNA polymerase (Pol)ε exhibits a highly elevated bias for forming T:dTTP mispairs over A:dATP mispairs and that yeast cells harboring this Polε mutation accumulate A > T signature mutations in the leading strand have been used to assign a role for Polε in replicating the leading strand. Here, we determine whether A > T signature mutations result from defects in Polε proofreading activity by analyzing their rate in Polε proofreading defective pol2-4 and pol2-M644G cells. Since purified pol2-4 Polε exhibits no bias for T:dTTP mispair formation, A > T mutations are expected to occur at a much lower rate in pol2-4 than in pol2-M644G cells if Polε replicated the leading strand. Instead, we find that the rate of A > T signature mutations are as highly elevated in pol2-4 cells as in pol2-M644G cells; furthermore, the highly elevated rate of A > T signature mutations is severely curtailed in the absence of PCNA ubiquitination or Polζ in both the pol2-M644G and pol2-4 strains. Altogether, our evidence supports the conclusion that the leading strand A > T signature mutations derive from defects in Polε proofreading activity and not from the role of Polε as a leading strand replicase, and it conforms with the genetic evidence for a major role of Polδ in replication of both the DNA strands.
KW - DNA polymerase δ
KW - DNA polymerase ε
KW - DNA polymerase ε defects in proofreading
KW - DNA polymerase ε role
KW - DNA polymerase ε signature mutations
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U2 - 10.1016/j.jbc.2023.104913
DO - 10.1016/j.jbc.2023.104913
M3 - Article
C2 - 37307920
AN - SCOPUS:85165003435
SN - 0021-9258
VL - 299
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 7
M1 - 104913
ER -