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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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The R137Q Pol β variant has an impaired interaction with PCNA. (A) Ni-NTA pull-down was performed with the cell extracts from pol β  MEFs and purified Pol β or R137Q variant proteins (His-tagged). The interaction was detected by western blotting using antibodies against FEN1, APE1, PCNA, Ligase 1, Ligase IIIα and XRCC1. A negative control with His-tagged GST was added to rule out the possibility that desirable proteins interact with His tag directly. (B) Pull-down assay using purified proteins. Purified PCNA was immobilized on Sepharose 4B beads, incubated with purified Pol β (WT or R137Q), washed and beads were then boiled in SDS–PAGE sample buffer. Proteins were separated by SDS–PAGE and transferred onto PVDF film, followed by detection with anti-Pol β and anti-PCNA antibodies. (C) Affinity comparison of PCNA with WT and R137Q Pol β by ELISA. PCNA was coated onto ELISA plates, incubated with different concentrations of purified WT or R137Q Pol β, washed and bound protein was detected by mouse anti-Pol β antibody and goat anti-mouse IgG/HRP. Color was developed by adding TMB. The color reaction was stopped by adding 1 N HCl. Optical density was read on microplate reader.
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Figure 3: The R137Q Pol β variant has an impaired interaction with PCNA. (A) Ni-NTA pull-down was performed with the cell extracts from pol β MEFs and purified Pol β or R137Q variant proteins (His-tagged). The interaction was detected by western blotting using antibodies against FEN1, APE1, PCNA, Ligase 1, Ligase IIIα and XRCC1. A negative control with His-tagged GST was added to rule out the possibility that desirable proteins interact with His tag directly. (B) Pull-down assay using purified proteins. Purified PCNA was immobilized on Sepharose 4B beads, incubated with purified Pol β (WT or R137Q), washed and beads were then boiled in SDS–PAGE sample buffer. Proteins were separated by SDS–PAGE and transferred onto PVDF film, followed by detection with anti-Pol β and anti-PCNA antibodies. (C) Affinity comparison of PCNA with WT and R137Q Pol β by ELISA. PCNA was coated onto ELISA plates, incubated with different concentrations of purified WT or R137Q Pol β, washed and bound protein was detected by mouse anti-Pol β antibody and goat anti-mouse IgG/HRP. Color was developed by adding TMB. The color reaction was stopped by adding 1 N HCl. Optical density was read on microplate reader.

Mentions: We then investigated the effects of R137Q substitution on protein–protein interactions. Extracts of pol β−/− MEF were incubated with purified WT or R137Q proteins (His-tagged) prior to immunoprecipitation of proteins involved in the BER pathway and detection by corresponding antibodies (Figure 3). Six proteins (APE1, FEN1, PCNA, XRCC1, Ligase I and Ligase IIIα) that interact with Pol β and are involved in either the SP- or LP-BER pathways were investigated. The amount of APE1, FEN1, XRCC1, Ligase 1 or Ligase IIIα that co-pulled down with R137Q was similar to that with the WT enzyme. However, there was a dramatic decrease in levels of R137Q-bound PCNA compared with WT Pol β-bound PCNA (Figure 3A, panel 2). To rule out the possibility that the His tag itself is responsible for the observed binding, we included His-tagged glutathione S-transferase (GST) as a control. In this case, none of the tested proteins binds to His-tag or GST. To further confirm this observation, we immobilized purified PCNA on Sepharose 4B beads and incubated them with purified WT or R137Q Pol β. Western blot of Sepharose bead-bound proteins showed that WT Pol β but not R137Q was efficiently pulled down by PCNA (Figure 3B), suggesting that the Arg137 to Gln substitution impaired the interaction between Pol β and PCNA. To quantitatively evaluate the impact of the R137Q substitution on the Pol β/PCNA interaction, we determined the relative amount of WT Pol β or R137Q-bound PCNA by ELISA. Our data suggested that levels of WT Pol β-bound PCNA were 1.5- to 4-fold higher than those of R137Q-bound PCNA (Figure 3C).Figure 3.


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)

The R137Q Pol β variant has an impaired interaction with PCNA. (A) Ni-NTA pull-down was performed with the cell extracts from pol β  MEFs and purified Pol β or R137Q variant proteins (His-tagged). The interaction was detected by western blotting using antibodies against FEN1, APE1, PCNA, Ligase 1, Ligase IIIα and XRCC1. A negative control with His-tagged GST was added to rule out the possibility that desirable proteins interact with His tag directly. (B) Pull-down assay using purified proteins. Purified PCNA was immobilized on Sepharose 4B beads, incubated with purified Pol β (WT or R137Q), washed and beads were then boiled in SDS–PAGE sample buffer. Proteins were separated by SDS–PAGE and transferred onto PVDF film, followed by detection with anti-Pol β and anti-PCNA antibodies. (C) Affinity comparison of PCNA with WT and R137Q Pol β by ELISA. PCNA was coated onto ELISA plates, incubated with different concentrations of purified WT or R137Q Pol β, washed and bound protein was detected by mouse anti-Pol β antibody and goat anti-mouse IgG/HRP. Color was developed by adding TMB. The color reaction was stopped by adding 1 N HCl. Optical density was read on microplate reader.
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Related In: Results  -  Collection

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Figure 3: The R137Q Pol β variant has an impaired interaction with PCNA. (A) Ni-NTA pull-down was performed with the cell extracts from pol β MEFs and purified Pol β or R137Q variant proteins (His-tagged). The interaction was detected by western blotting using antibodies against FEN1, APE1, PCNA, Ligase 1, Ligase IIIα and XRCC1. A negative control with His-tagged GST was added to rule out the possibility that desirable proteins interact with His tag directly. (B) Pull-down assay using purified proteins. Purified PCNA was immobilized on Sepharose 4B beads, incubated with purified Pol β (WT or R137Q), washed and beads were then boiled in SDS–PAGE sample buffer. Proteins were separated by SDS–PAGE and transferred onto PVDF film, followed by detection with anti-Pol β and anti-PCNA antibodies. (C) Affinity comparison of PCNA with WT and R137Q Pol β by ELISA. PCNA was coated onto ELISA plates, incubated with different concentrations of purified WT or R137Q Pol β, washed and bound protein was detected by mouse anti-Pol β antibody and goat anti-mouse IgG/HRP. Color was developed by adding TMB. The color reaction was stopped by adding 1 N HCl. Optical density was read on microplate reader.
Mentions: We then investigated the effects of R137Q substitution on protein–protein interactions. Extracts of pol β−/− MEF were incubated with purified WT or R137Q proteins (His-tagged) prior to immunoprecipitation of proteins involved in the BER pathway and detection by corresponding antibodies (Figure 3). Six proteins (APE1, FEN1, PCNA, XRCC1, Ligase I and Ligase IIIα) that interact with Pol β and are involved in either the SP- or LP-BER pathways were investigated. The amount of APE1, FEN1, XRCC1, Ligase 1 or Ligase IIIα that co-pulled down with R137Q was similar to that with the WT enzyme. However, there was a dramatic decrease in levels of R137Q-bound PCNA compared with WT Pol β-bound PCNA (Figure 3A, panel 2). To rule out the possibility that the His tag itself is responsible for the observed binding, we included His-tagged glutathione S-transferase (GST) as a control. In this case, none of the tested proteins binds to His-tag or GST. To further confirm this observation, we immobilized purified PCNA on Sepharose 4B beads and incubated them with purified WT or R137Q Pol β. Western blot of Sepharose bead-bound proteins showed that WT Pol β but not R137Q was efficiently pulled down by PCNA (Figure 3B), suggesting that the Arg137 to Gln substitution impaired the interaction between Pol β and PCNA. To quantitatively evaluate the impact of the R137Q substitution on the Pol β/PCNA interaction, we determined the relative amount of WT Pol β or R137Q-bound PCNA by ELISA. Our data suggested that levels of WT Pol β-bound PCNA were 1.5- to 4-fold higher than those of R137Q-bound PCNA (Figure 3C).Figure 3.

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