Limits...
Epistatic role of base excision repair and mismatch repair pathways in mediating cisplatin cytotoxicity.

Kothandapani A, Sawant A, Dangeti VS, Sobol RW, Patrick SM - Nucleic Acids Res. (2013)

Bottom Line: MSH2 preferentially binds a cisplatin interstrand cross-link (ICL) DNA substrate containing a mismatch compared with a cisplatin ICL substrate without a mismatch, suggesting a novel mutagenic role of Polβ in activating MMR in response to cisplatin.Collectively, these results provide the first mechanistic model for BER and MMR functioning within the same pathway to mediate cisplatin sensitivity via non-productive ICL processing.In this model, MMR participation in non-productive cisplatin ICL processing is downstream of BER processing and dependent on Polβ misincorporation at cisplatin ICL sites, which results in persistent cisplatin ICLs and sensitivity to cisplatin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cancer Biology, University of Toledo - Health Science Campus, Toledo, OH 43614, USA, Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA, University of Pittsburgh Cancer Institute, Hillman Cancer Center, Pittsburgh, PA 15213, USA and Department of Human Genetics, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA 15213, USA.

ABSTRACT
Base excision repair (BER) and mismatch repair (MMR) pathways play an important role in modulating cis-Diamminedichloroplatinum (II) (cisplatin) cytotoxicity. In this article, we identified a novel mechanistic role of both BER and MMR pathways in mediating cellular responses to cisplatin treatment. Cells defective in BER or MMR display a cisplatin-resistant phenotype. Targeting both BER and MMR pathways resulted in no additional resistance to cisplatin, suggesting that BER and MMR play epistatic roles in mediating cisplatin cytotoxicity. Using a DNA Polymerase β (Polβ) variant deficient in polymerase activity (D256A), we demonstrate that MMR acts downstream of BER and is dependent on the polymerase activity of Polβ in mediating cisplatin cytotoxicity. MSH2 preferentially binds a cisplatin interstrand cross-link (ICL) DNA substrate containing a mismatch compared with a cisplatin ICL substrate without a mismatch, suggesting a novel mutagenic role of Polβ in activating MMR in response to cisplatin. Collectively, these results provide the first mechanistic model for BER and MMR functioning within the same pathway to mediate cisplatin sensitivity via non-productive ICL processing. In this model, MMR participation in non-productive cisplatin ICL processing is downstream of BER processing and dependent on Polβ misincorporation at cisplatin ICL sites, which results in persistent cisplatin ICLs and sensitivity to cisplatin.

Show MeSH

Related in: MedlinePlus

Interaction of MSH2 with cisplatin ICL DNA containing a mismatch. (A) Undamaged and single cisplatin ICL containing duplexes with and without a mismatch were bound to streptavidin magnetic beads and were incubated with equal amounts of overexpressed hMSH2-hMSH6 insect cell extract. The proteins bound to the DNA beads were eluted and immunoblotted with the antibody against MSH2. (B) EMSAs of hMSH2-hMSH6 binding to undamaged duplex DNA, G/T mismatch, cisplatin ICL and cisplatin ICL G/T substrates. (C) Duplex biotinylated 42 mers containing a uracil in undamaged DNA substrates or adjacent to a cisplatin ICL were processed with UDG, Ape1 and Polβ before carrying out similar pull-down assays.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3753620&req=5

gkt479-F5: Interaction of MSH2 with cisplatin ICL DNA containing a mismatch. (A) Undamaged and single cisplatin ICL containing duplexes with and without a mismatch were bound to streptavidin magnetic beads and were incubated with equal amounts of overexpressed hMSH2-hMSH6 insect cell extract. The proteins bound to the DNA beads were eluted and immunoblotted with the antibody against MSH2. (B) EMSAs of hMSH2-hMSH6 binding to undamaged duplex DNA, G/T mismatch, cisplatin ICL and cisplatin ICL G/T substrates. (C) Duplex biotinylated 42 mers containing a uracil in undamaged DNA substrates or adjacent to a cisplatin ICL were processed with UDG, Ape1 and Polβ before carrying out similar pull-down assays.

Mentions: Biotinylated duplex DNAs either undamaged or containing a single mismatch, a single cisplatin ICL or a cisplatin ICL with a mismatch were synthesized as described previously (30,35). The substrates were bound to streptavidin magnetic beads (Dynabeads, Dynal Biotech) in the presence of binding buffer [20 mM HEPES (pH 7.8), 2 mM DTT, 0.001% NP-40, 100 mM NaCl and 200 mM MgCl2] at 4°C for 30 min. The beads were washed three times with binding buffer to remove all the unbound DNA substrates. Next, 5 µg of hMSH2-hMSH6 baculovirus infected SF-9 insect cell extract was added with 30-fold excess of poly dI-dC competitor DNA. The tubes were rotated for 1 h at 4°C. All the tubes were kept in a magnetic separation stand (Promega). The tubes were then washed three times in wash buffer (binding buffer with 200 mM NaCl), followed by elution using 1M NaCl. The tubes were left to rotate for 30 min and again placed in a magnetic separation stand, and the supernatants were collected and trichloroacetic acid (TCA) precipitated. The resulting pellets were resuspended in binding buffer and loaded onto 8% SDS gels, transferred to PVDF membrane and probed with MSH2 (Calbiochem) antibody. A similar experimental protocol was followed for the undamaged and cisplatin ICL substrate containing a uracil adjacent to the cross-link except that it was treated with uracil DNA glycosylase (UDG), Apurinic endonuclease (Ape1) and DNA Polβ in the presence of all the dNTPs. This BER processed DNA generates a substrate with either a correct or incorrect base adjacent to the cisplatin ICL (30). The undamaged and cisplatin ICL DNA substrates were recovered by ethanol precipitation and used for the Biotin–DNA–streptavidin pull down experiments in Figure 5C.


Epistatic role of base excision repair and mismatch repair pathways in mediating cisplatin cytotoxicity.

Kothandapani A, Sawant A, Dangeti VS, Sobol RW, Patrick SM - Nucleic Acids Res. (2013)

Interaction of MSH2 with cisplatin ICL DNA containing a mismatch. (A) Undamaged and single cisplatin ICL containing duplexes with and without a mismatch were bound to streptavidin magnetic beads and were incubated with equal amounts of overexpressed hMSH2-hMSH6 insect cell extract. The proteins bound to the DNA beads were eluted and immunoblotted with the antibody against MSH2. (B) EMSAs of hMSH2-hMSH6 binding to undamaged duplex DNA, G/T mismatch, cisplatin ICL and cisplatin ICL G/T substrates. (C) Duplex biotinylated 42 mers containing a uracil in undamaged DNA substrates or adjacent to a cisplatin ICL were processed with UDG, Ape1 and Polβ before carrying out similar pull-down assays.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3753620&req=5

gkt479-F5: Interaction of MSH2 with cisplatin ICL DNA containing a mismatch. (A) Undamaged and single cisplatin ICL containing duplexes with and without a mismatch were bound to streptavidin magnetic beads and were incubated with equal amounts of overexpressed hMSH2-hMSH6 insect cell extract. The proteins bound to the DNA beads were eluted and immunoblotted with the antibody against MSH2. (B) EMSAs of hMSH2-hMSH6 binding to undamaged duplex DNA, G/T mismatch, cisplatin ICL and cisplatin ICL G/T substrates. (C) Duplex biotinylated 42 mers containing a uracil in undamaged DNA substrates or adjacent to a cisplatin ICL were processed with UDG, Ape1 and Polβ before carrying out similar pull-down assays.
Mentions: Biotinylated duplex DNAs either undamaged or containing a single mismatch, a single cisplatin ICL or a cisplatin ICL with a mismatch were synthesized as described previously (30,35). The substrates were bound to streptavidin magnetic beads (Dynabeads, Dynal Biotech) in the presence of binding buffer [20 mM HEPES (pH 7.8), 2 mM DTT, 0.001% NP-40, 100 mM NaCl and 200 mM MgCl2] at 4°C for 30 min. The beads were washed three times with binding buffer to remove all the unbound DNA substrates. Next, 5 µg of hMSH2-hMSH6 baculovirus infected SF-9 insect cell extract was added with 30-fold excess of poly dI-dC competitor DNA. The tubes were rotated for 1 h at 4°C. All the tubes were kept in a magnetic separation stand (Promega). The tubes were then washed three times in wash buffer (binding buffer with 200 mM NaCl), followed by elution using 1M NaCl. The tubes were left to rotate for 30 min and again placed in a magnetic separation stand, and the supernatants were collected and trichloroacetic acid (TCA) precipitated. The resulting pellets were resuspended in binding buffer and loaded onto 8% SDS gels, transferred to PVDF membrane and probed with MSH2 (Calbiochem) antibody. A similar experimental protocol was followed for the undamaged and cisplatin ICL substrate containing a uracil adjacent to the cross-link except that it was treated with uracil DNA glycosylase (UDG), Apurinic endonuclease (Ape1) and DNA Polβ in the presence of all the dNTPs. This BER processed DNA generates a substrate with either a correct or incorrect base adjacent to the cisplatin ICL (30). The undamaged and cisplatin ICL DNA substrates were recovered by ethanol precipitation and used for the Biotin–DNA–streptavidin pull down experiments in Figure 5C.

Bottom Line: MSH2 preferentially binds a cisplatin interstrand cross-link (ICL) DNA substrate containing a mismatch compared with a cisplatin ICL substrate without a mismatch, suggesting a novel mutagenic role of Polβ in activating MMR in response to cisplatin.Collectively, these results provide the first mechanistic model for BER and MMR functioning within the same pathway to mediate cisplatin sensitivity via non-productive ICL processing.In this model, MMR participation in non-productive cisplatin ICL processing is downstream of BER processing and dependent on Polβ misincorporation at cisplatin ICL sites, which results in persistent cisplatin ICLs and sensitivity to cisplatin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cancer Biology, University of Toledo - Health Science Campus, Toledo, OH 43614, USA, Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA, University of Pittsburgh Cancer Institute, Hillman Cancer Center, Pittsburgh, PA 15213, USA and Department of Human Genetics, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA 15213, USA.

ABSTRACT
Base excision repair (BER) and mismatch repair (MMR) pathways play an important role in modulating cis-Diamminedichloroplatinum (II) (cisplatin) cytotoxicity. In this article, we identified a novel mechanistic role of both BER and MMR pathways in mediating cellular responses to cisplatin treatment. Cells defective in BER or MMR display a cisplatin-resistant phenotype. Targeting both BER and MMR pathways resulted in no additional resistance to cisplatin, suggesting that BER and MMR play epistatic roles in mediating cisplatin cytotoxicity. Using a DNA Polymerase β (Polβ) variant deficient in polymerase activity (D256A), we demonstrate that MMR acts downstream of BER and is dependent on the polymerase activity of Polβ in mediating cisplatin cytotoxicity. MSH2 preferentially binds a cisplatin interstrand cross-link (ICL) DNA substrate containing a mismatch compared with a cisplatin ICL substrate without a mismatch, suggesting a novel mutagenic role of Polβ in activating MMR in response to cisplatin. Collectively, these results provide the first mechanistic model for BER and MMR functioning within the same pathway to mediate cisplatin sensitivity via non-productive ICL processing. In this model, MMR participation in non-productive cisplatin ICL processing is downstream of BER processing and dependent on Polβ misincorporation at cisplatin ICL sites, which results in persistent cisplatin ICLs and sensitivity to cisplatin.

Show MeSH
Related in: MedlinePlus