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Human DNA polymerase beta polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity.

Guo Z, Zheng L, Dai H, Zhou M, Xu H, Shen B - Nucleic Acids Res. (2009)

Bottom Line: More than 30% of human tumors characterized to date express DNA Pol beta variants, many of which result from a single nucleotide residue substitution.The substitution also affects the interaction between Pol beta and proliferating cell nuclear antigen (PCNA).Expression of wild-type Pol beta in pol beta(-/-) mouse embryonic fibroblast (MEF) cells restored cellular resistance to DNA damaging reagents such as methyl methanesulfonate (MMS) and N-methyl-N-nitrosourea (MNU), while expression of R137Q in pol beta(-/-) MEF cells failed to do so.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Biology, City of Hope National Medical Center, Beckman Research Institute, Duarte, CA 91010, USA.

ABSTRACT
DNA polymerase beta (Pol beta) is a key enzyme in DNA base excision repair, and an important factor for maintaining genome integrity and stability. More than 30% of human tumors characterized to date express DNA Pol beta variants, many of which result from a single nucleotide residue substitution. However, in most cases, their precise functional deficiency and relationship to cancer susceptibility are still unknown. In the current work, we show that a polymorphism encoding an arginine to glutamine substitution, R137Q, has lower polymerase activity. The substitution also affects the interaction between Pol beta and proliferating cell nuclear antigen (PCNA). These defects impair the DNA repair capacity of Pol beta in reconstitution assays, as well as in cellular extracts. Expression of wild-type Pol beta in pol beta(-/-) mouse embryonic fibroblast (MEF) cells restored cellular resistance to DNA damaging reagents such as methyl methanesulfonate (MMS) and N-methyl-N-nitrosourea (MNU), while expression of R137Q in pol beta(-/-) MEF cells failed to do so. These data indicate that polymorphisms in base excision repair genes may contribute to the onset and development of cancers.

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Cells harboring polymorphism R137Q are sensitive to DNA damage stress. (A) Abasic-specific DNA damage assay. Cells were treated with MMS. The amount of damaged DNA lesions was then detected by ARP reagent (see Experimental Procedures section). (B) Effect of MMS on induction of apoptosis in complemented pol β−/− MEFs. Pol β−/− MEFs complemented with WT or the R137Q variant of Pol β were treated (1 h) with 2 mM MMS. Adherent and floating cells were harvested after 24 h and analyzed by flow cytometry. The percentage of the apoptotic cells indicated. The MMS-induced increase of apoptotic cells is shown in the bottom bar. WT, pol β−/−, pol β−/−/WT and pol β−/−/R137Q MEF cells were treated (1 h) with MMS (C) and MNU (D) at the indicated concentrations. Cellular sensitivity was determined by growth inhibition experiments. Data represents mean ± SD of four independent experiments. WT, filled squares; pol β−/−/WT, filled triangles; pol β−/−, filled circles; pol β−/−/R137Q, open squares.
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Figure 6: Cells harboring polymorphism R137Q are sensitive to DNA damage stress. (A) Abasic-specific DNA damage assay. Cells were treated with MMS. The amount of damaged DNA lesions was then detected by ARP reagent (see Experimental Procedures section). (B) Effect of MMS on induction of apoptosis in complemented pol β−/− MEFs. Pol β−/− MEFs complemented with WT or the R137Q variant of Pol β were treated (1 h) with 2 mM MMS. Adherent and floating cells were harvested after 24 h and analyzed by flow cytometry. The percentage of the apoptotic cells indicated. The MMS-induced increase of apoptotic cells is shown in the bottom bar. WT, pol β−/−, pol β−/−/WT and pol β−/−/R137Q MEF cells were treated (1 h) with MMS (C) and MNU (D) at the indicated concentrations. Cellular sensitivity was determined by growth inhibition experiments. Data represents mean ± SD of four independent experiments. WT, filled squares; pol β−/−/WT, filled triangles; pol β−/−, filled circles; pol β−/−/R137Q, open squares.

Mentions: Our observation that R137Q Pol β caused defective BER led us to propose that cells expressing the R137Q variant would be sensitive to DNA base damaging agents. To test this hypothesis, MEF cells of WT, pol β knockout (pol β−/−) and pol β−/− expressing human WT or R137Q Pol β (designated as pol β−/−/WT or pol−/−/R137Q) were treated with MMS. Consistent with a previous report (15), we found that deletion of pol β caused cells to accumulate MMS-induced DNA damage at MMS concentrations ≥1 mM (Figure 6A) and to become sensitive to the MMS and MNU (Figure 6B–D). While human WT Pol β fully complemented the function of its mouse homolog, R137Q only partially complemented it. FACS data showed that apoptotic cells of pol β knock out or R137Q genetic background were considerably higher than the WT background. Additionally, there was a 5-fold increase in MMS-induced apoptotic R137Q cells compared with WT (3.1% versus 0.6%) (Figure 6B). MMS consistently had a stronger inhibitory effect on pol β−/−-R137Q cell growth relative to WT or pol β−/−/WT cells. Following MMS or MNU treatment, the ratio of MMS or MNU treated/untreated pol β−/−-R137Q cells was <10%, whereas that for WT or pol β−/−-WT cells was >90% (Figure 6C and D).Figure 6.


Human DNA polymerase beta polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity.

Guo Z, Zheng L, Dai H, Zhou M, Xu H, Shen B - Nucleic Acids Res. (2009)

Cells harboring polymorphism R137Q are sensitive to DNA damage stress. (A) Abasic-specific DNA damage assay. Cells were treated with MMS. The amount of damaged DNA lesions was then detected by ARP reagent (see Experimental Procedures section). (B) Effect of MMS on induction of apoptosis in complemented pol β−/− MEFs. Pol β−/− MEFs complemented with WT or the R137Q variant of Pol β were treated (1 h) with 2 mM MMS. Adherent and floating cells were harvested after 24 h and analyzed by flow cytometry. The percentage of the apoptotic cells indicated. The MMS-induced increase of apoptotic cells is shown in the bottom bar. WT, pol β−/−, pol β−/−/WT and pol β−/−/R137Q MEF cells were treated (1 h) with MMS (C) and MNU (D) at the indicated concentrations. Cellular sensitivity was determined by growth inhibition experiments. Data represents mean ± SD of four independent experiments. WT, filled squares; pol β−/−/WT, filled triangles; pol β−/−, filled circles; pol β−/−/R137Q, open squares.
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Related In: Results  -  Collection

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Figure 6: Cells harboring polymorphism R137Q are sensitive to DNA damage stress. (A) Abasic-specific DNA damage assay. Cells were treated with MMS. The amount of damaged DNA lesions was then detected by ARP reagent (see Experimental Procedures section). (B) Effect of MMS on induction of apoptosis in complemented pol β−/− MEFs. Pol β−/− MEFs complemented with WT or the R137Q variant of Pol β were treated (1 h) with 2 mM MMS. Adherent and floating cells were harvested after 24 h and analyzed by flow cytometry. The percentage of the apoptotic cells indicated. The MMS-induced increase of apoptotic cells is shown in the bottom bar. WT, pol β−/−, pol β−/−/WT and pol β−/−/R137Q MEF cells were treated (1 h) with MMS (C) and MNU (D) at the indicated concentrations. Cellular sensitivity was determined by growth inhibition experiments. Data represents mean ± SD of four independent experiments. WT, filled squares; pol β−/−/WT, filled triangles; pol β−/−, filled circles; pol β−/−/R137Q, open squares.
Mentions: Our observation that R137Q Pol β caused defective BER led us to propose that cells expressing the R137Q variant would be sensitive to DNA base damaging agents. To test this hypothesis, MEF cells of WT, pol β knockout (pol β−/−) and pol β−/− expressing human WT or R137Q Pol β (designated as pol β−/−/WT or pol−/−/R137Q) were treated with MMS. Consistent with a previous report (15), we found that deletion of pol β caused cells to accumulate MMS-induced DNA damage at MMS concentrations ≥1 mM (Figure 6A) and to become sensitive to the MMS and MNU (Figure 6B–D). While human WT Pol β fully complemented the function of its mouse homolog, R137Q only partially complemented it. FACS data showed that apoptotic cells of pol β knock out or R137Q genetic background were considerably higher than the WT background. Additionally, there was a 5-fold increase in MMS-induced apoptotic R137Q cells compared with WT (3.1% versus 0.6%) (Figure 6B). MMS consistently had a stronger inhibitory effect on pol β−/−-R137Q cell growth relative to WT or pol β−/−/WT cells. Following MMS or MNU treatment, the ratio of MMS or MNU treated/untreated pol β−/−-R137Q cells was <10%, whereas that for WT or pol β−/−-WT cells was >90% (Figure 6C and D).Figure 6.

Bottom Line: More than 30% of human tumors characterized to date express DNA Pol beta variants, many of which result from a single nucleotide residue substitution.The substitution also affects the interaction between Pol beta and proliferating cell nuclear antigen (PCNA).Expression of wild-type Pol beta in pol beta(-/-) mouse embryonic fibroblast (MEF) cells restored cellular resistance to DNA damaging reagents such as methyl methanesulfonate (MMS) and N-methyl-N-nitrosourea (MNU), while expression of R137Q in pol beta(-/-) MEF cells failed to do so.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Biology, City of Hope National Medical Center, Beckman Research Institute, Duarte, CA 91010, USA.

ABSTRACT
DNA polymerase beta (Pol beta) is a key enzyme in DNA base excision repair, and an important factor for maintaining genome integrity and stability. More than 30% of human tumors characterized to date express DNA Pol beta variants, many of which result from a single nucleotide residue substitution. However, in most cases, their precise functional deficiency and relationship to cancer susceptibility are still unknown. In the current work, we show that a polymorphism encoding an arginine to glutamine substitution, R137Q, has lower polymerase activity. The substitution also affects the interaction between Pol beta and proliferating cell nuclear antigen (PCNA). These defects impair the DNA repair capacity of Pol beta in reconstitution assays, as well as in cellular extracts. Expression of wild-type Pol beta in pol beta(-/-) mouse embryonic fibroblast (MEF) cells restored cellular resistance to DNA damaging reagents such as methyl methanesulfonate (MMS) and N-methyl-N-nitrosourea (MNU), while expression of R137Q in pol beta(-/-) MEF cells failed to do so. These data indicate that polymorphisms in base excision repair genes may contribute to the onset and development of cancers.

Show MeSH
Related in: MedlinePlus