AP endonuclease knockdown enhances methyl methanesulfonate hypersensitivity of DNA polymerase β knockout mouse embryonic fibroblasts.
Apurinic/apyrimidinic (AP) endonuclease (Apex) is required for base excision repair (BER), which is the major mechanism of repair for small DNA lesions such as alkylated bases. Apex incises the DNA strand at an AP site to leave 3'-OH and 5'-deoxyribose phosphate (5'-dRp) termini. DNA polymerase β (PolB) plays a dominant role in single nucleotide (Sn-) BER by incorporating a nucleotide and removing 5'-dRp. Methyl methanesulfonate (MMS)-induced damage is repaired by Sn-BER, and thus mouse embryonic fibroblasts (MEFs) deficient in PolB show significantly increased sensitivity to MMS. However, the survival curve for PolB-knockout MEFs (PolBKOs) has a shoulder, and increased sensitivity is only apparent at relatively high MMS concentrations. In this study, we prepared Apex-knockdown/PolB-knockout MEFs (AKDBKOs) to examine whether BER is related to the apparent resistance of PolBKOs at low MMS concentrations. The viability of PolBKOs immediately after MMS treatment was significantly lower than that of wild-type MEFs, but there was essentially no effect of Apex-knockdown on cell viability in the presence or absence of PolB. In contrast, relative counts of MEFs after repair were decreased by Apex knockdown. Parental PolBKOs showed especially high sensitivity at >1.5 mM MMS, suggesting that PolBKOs have another repair mechanism in addition to PolB-dependent Sn-BER, and that the back-up mechanism is unable to repair damage induced by high MMS concentrations. Interestingly, AKDBKOs were hypersensitive to MMS in a relative cell growth assay, suggesting that MMS-induced damage in PolB-knockout MEFs is repaired by Apex-dependent repair mechanisms, presumably including long-patch BER.
Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku Ourai Kita, Izumisano, Osaka 598-8531, Japan yamamoto@vet.osakafu-u.ac.jp.
Ryohei
R
Yamamoto
Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku Ourai Kita, Izumisano, Osaka 598-8531, Japan.
Makio
M
Umetsu
Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku Ourai Kita, Izumisano, Osaka 598-8531, Japan.
Mizuki
M
Yamamoto
Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku Ourai Kita, Izumisano, Osaka 598-8531, Japan.
Satoshi
S
Matsuyama
Department of Integrated Functional Biosciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku Ourai Kita, Izumisano, Osaka 598-8531, Japan.
Shigeo
S
Takenaka
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan.
Hiroshi
H
Ide
Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku Ourai Kita, Izumisano, Osaka 598-8531, Japan.
Kihei
K
Kubo
D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D019951:DNA Polymerase beta / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair / Q000187:drug effects / Q000502:physiology*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D008741:Methyl Methanesulfonate / Q000008:administration & dosage*; D018345:Mice, Knockout; D009153:Mutagens / Q000008:administration & dosage
D009153:Mutagens; D008741:Methyl Methanesulfonate; D019951:DNA Polymerase beta; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase
Apex; BER; MEF; MMS; Polβ; mouse