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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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R137Q significantly reduces BER efficiency. (A) SP-BER reconstitution with purified WT and R137Q Pol β. (B) LP-BER reconstitution with purified WT and R137Q. The top part of each panel shows the schematic structures of the corresponding DNA substrates. The middle shows PAGE-separated products and the bottom the relative percentage of repaired product obtained with the indicated amounts of Pol β. Values represent mean ± SD of three independent assays. WT, filled squares; R137, filled circle.
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Figure 4: R137Q significantly reduces BER efficiency. (A) SP-BER reconstitution with purified WT and R137Q Pol β. (B) LP-BER reconstitution with purified WT and R137Q. The top part of each panel shows the schematic structures of the corresponding DNA substrates. The middle shows PAGE-separated products and the bottom the relative percentage of repaired product obtained with the indicated amounts of Pol β. Values represent mean ± SD of three independent assays. WT, filled squares; R137, filled circle.

Mentions: The impaired biochemical functions of the R137Q variant suggested that R137Q likely affected BER function. To test this hypothesis, SP- and LP-BER were assayed using purified proteins. Uracil-containing substrate (Pol β-U) and tetrahydrofuran-containing substrate (Pol β-F) were used for SP- and LP-BER substrates, respectively (Table 1). Cleavage of the uracil or Tetrahydrofuran (THF) lesion by the concerted action of UDG and APE1 resulted in a nicked DNA duplex, and incorporation of 32P-dCTP and other deoxynucleotides produced 20–30 nt non-ligated intermediates visible to the phosphorimager (Figure 4A and B). Further processing of this intermediate structure generated a fully repaired product of 40 nt. We observed that the uracil or THF lesions were efficiently repaired in the presence of WT Pol β but not the R137Q variant (Figure 4A and B). The reduced repair efficiency by R137Q was likely due to its low-polymerase activity. Consistent with the polymerase activity assays (Figure 1A), only a small amount of 32P-dCTP was incorporated into the DNA substrate by R137Q in reconstituted SP- and LP-BER reactions (Figure 4A and B). To validate that the R137Q variant lead to low-BER efficiency, we expressed human WT and R137Q Pol β in mouse MEF of pol β knockout genetic background. Cells expressing similar level of Pol β were selected (Figure 5A). Nuclear extracts (NEs) from these cell lines were prepared and their BER efficiency assayed. We found that WT NE efficiently repaired the uracil or THF lesion, resulting in a 40 nt band, whereas the repair efficiency by R137Q NE was only ∼10 and 20% for SP- and LP-BER, respectively (Figure 5B and C).Figure 4.


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)

R137Q significantly reduces BER efficiency. (A) SP-BER reconstitution with purified WT and R137Q Pol β. (B) LP-BER reconstitution with purified WT and R137Q. The top part of each panel shows the schematic structures of the corresponding DNA substrates. The middle shows PAGE-separated products and the bottom the relative percentage of repaired product obtained with the indicated amounts of Pol β. Values represent mean ± SD of three independent assays. WT, filled squares; R137, filled circle.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2691839&req=5

Figure 4: R137Q significantly reduces BER efficiency. (A) SP-BER reconstitution with purified WT and R137Q Pol β. (B) LP-BER reconstitution with purified WT and R137Q. The top part of each panel shows the schematic structures of the corresponding DNA substrates. The middle shows PAGE-separated products and the bottom the relative percentage of repaired product obtained with the indicated amounts of Pol β. Values represent mean ± SD of three independent assays. WT, filled squares; R137, filled circle.
Mentions: The impaired biochemical functions of the R137Q variant suggested that R137Q likely affected BER function. To test this hypothesis, SP- and LP-BER were assayed using purified proteins. Uracil-containing substrate (Pol β-U) and tetrahydrofuran-containing substrate (Pol β-F) were used for SP- and LP-BER substrates, respectively (Table 1). Cleavage of the uracil or Tetrahydrofuran (THF) lesion by the concerted action of UDG and APE1 resulted in a nicked DNA duplex, and incorporation of 32P-dCTP and other deoxynucleotides produced 20–30 nt non-ligated intermediates visible to the phosphorimager (Figure 4A and B). Further processing of this intermediate structure generated a fully repaired product of 40 nt. We observed that the uracil or THF lesions were efficiently repaired in the presence of WT Pol β but not the R137Q variant (Figure 4A and B). The reduced repair efficiency by R137Q was likely due to its low-polymerase activity. Consistent with the polymerase activity assays (Figure 1A), only a small amount of 32P-dCTP was incorporated into the DNA substrate by R137Q in reconstituted SP- and LP-BER reactions (Figure 4A and B). To validate that the R137Q variant lead to low-BER efficiency, we expressed human WT and R137Q Pol β in mouse MEF of pol β knockout genetic background. Cells expressing similar level of Pol β were selected (Figure 5A). Nuclear extracts (NEs) from these cell lines were prepared and their BER efficiency assayed. We found that WT NE efficiently repaired the uracil or THF lesion, resulting in a 40 nt band, whereas the repair efficiency by R137Q NE was only ∼10 and 20% for SP- and LP-BER, respectively (Figure 5B and C).Figure 4.

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