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Uracil-DNA glycosylases SMUG1 and UNG2 coordinate the initial steps of base excision repair by distinct mechanisms.

Pettersen HS, Sundheim O, Gilljam KM, Slupphaug G, Krokan HE, Kavli B - Nucleic Acids Res. (2007)

Bottom Line: Mutations in this motif increase catalytic turnover due to reduced product binding.In contrast, the highly efficient UNG2 lacks product-binding capacity and stimulates AP-site cleavage by APE1, facilitating the two first steps in BER.In summary, this work reveals that SMUG1 and UNG2 coordinate the initial steps of BER by distinct mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Research and Molecular Medicine, NTNU, N-7006 Trondheim, Norway.

ABSTRACT
DNA glycosylases UNG and SMUG1 excise uracil from DNA and belong to the same protein superfamily. Vertebrates contain both SMUG1 and UNG, but their distinct roles in base excision repair (BER) of deaminated cytosine (U:G) are still not fully defined. Here we have examined the ability of human SMUG1 and UNG2 (nuclear UNG) to initiate and coordinate repair of U:G mismatches. When expressed in Escherichia coli cells, human UNG2 initiates complete repair of deaminated cytosine, while SMUG1 inhibits cell proliferation. In vitro, we show that SMUG1 binds tightly to AP-sites and inhibits AP-site cleavage by AP-endonucleases. Furthermore, a specific motif important for the AP-site product binding has been identified. Mutations in this motif increase catalytic turnover due to reduced product binding. In contrast, the highly efficient UNG2 lacks product-binding capacity and stimulates AP-site cleavage by APE1, facilitating the two first steps in BER. In summary, this work reveals that SMUG1 and UNG2 coordinate the initial steps of BER by distinct mechanisms. UNG2 is apparently adapted to rapid and highly coordinated repair of uracil (U:G and U:A) in replicating DNA, while the less efficient SMUG1 may be more important in repair of deaminated cytosine (U:G) in non-replicating chromatin.

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Product binding, effects of the AP-endonuclease APE1 and effects on AP-endonucleases APE1 and Exo III. (A) EMSA analysis of SMUG1 and UNG2 on AP-sites; 50, 100, 200, 300, 400 and 500 nM hSMUG1 and hUNG2 were incubated with 4 nM ds oligonucleotide (with complementary bases as indicated) or ss oligonucleotide, following uracil-excision binding to the product AP-sites were analysed by non-denaturating PAGE. (B) Percent bound AP:G and AP:A oligo plotted against the concentration of SMUG1 (µM) using a sigmoid curve fit model in GraphPad Prism®. The Kd values were calculated and represent the concentration of SMUG1 giving 50% of maximal binding to the AP-oligo. (C) Multiple turnover oligonucleotide assay with 0.85 nM SMUG1 and 20 nM U:G substrate (solid lines) or 20 nM Uss substrate (dotted lines) in the absence or presence of 8.5 nM APE1. (D) Multiple turnover oligonucleotide assay with 0.085 nM UNG2 and 20 nM U:G substrate (solid lines) or 20 nM Uss substrate (dotted lines) in the absence or presence of 8.5 nM APE1. Uracil excision was quantified after piperidine cleavage of the AP-site and separation by PAGE. (E) AP-cleavage activity of APE1 and ExoIII in the presence of SMUG1. (F) AP-cleavage activity of APE1 and ExoIII in the presence of UNG2. In (E) and (F), the AP-endonucleases (0.025 nM) were incubated for 10 min with 2 nM oligonucleotide substrate containing a central AP-site opposite guanine (AP:G) together with increasing amounts (0–1 µM) of SMUG1 or UNG2. AP-endonucleolytic cleavage was quantified after separation by PAGE.
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Figure 3: Product binding, effects of the AP-endonuclease APE1 and effects on AP-endonucleases APE1 and Exo III. (A) EMSA analysis of SMUG1 and UNG2 on AP-sites; 50, 100, 200, 300, 400 and 500 nM hSMUG1 and hUNG2 were incubated with 4 nM ds oligonucleotide (with complementary bases as indicated) or ss oligonucleotide, following uracil-excision binding to the product AP-sites were analysed by non-denaturating PAGE. (B) Percent bound AP:G and AP:A oligo plotted against the concentration of SMUG1 (µM) using a sigmoid curve fit model in GraphPad Prism®. The Kd values were calculated and represent the concentration of SMUG1 giving 50% of maximal binding to the AP-oligo. (C) Multiple turnover oligonucleotide assay with 0.85 nM SMUG1 and 20 nM U:G substrate (solid lines) or 20 nM Uss substrate (dotted lines) in the absence or presence of 8.5 nM APE1. (D) Multiple turnover oligonucleotide assay with 0.085 nM UNG2 and 20 nM U:G substrate (solid lines) or 20 nM Uss substrate (dotted lines) in the absence or presence of 8.5 nM APE1. Uracil excision was quantified after piperidine cleavage of the AP-site and separation by PAGE. (E) AP-cleavage activity of APE1 and ExoIII in the presence of SMUG1. (F) AP-cleavage activity of APE1 and ExoIII in the presence of UNG2. In (E) and (F), the AP-endonucleases (0.025 nM) were incubated for 10 min with 2 nM oligonucleotide substrate containing a central AP-site opposite guanine (AP:G) together with increasing amounts (0–1 µM) of SMUG1 or UNG2. AP-endonucleolytic cleavage was quantified after separation by PAGE.

Mentions: To elucidate the molecular mechanisms underlying the observed in vivo effects of SMUG1 and UNG2, we analysed the product (AP-site) binding subsequent to uracil-excision by purified human SMUG1 and UNG2 using electrophoretic mobility shift assays (EMSA). The results, illustrated in Figure 3A, demonstrate that SMUG1 readily binds to AP-sites in dsDNA (AP:G and AP:A), while no binding to AP-sites in single-stranded DNA (APss) or dsDNA without AP-site (T:A) was detected. In contrast, we did not observe binding of UNG2 to the same set of oligonucleotides. SMUG1 binds AP:G with slightly higher affinity than AP:A with Kd values (concentration of enzyme yielding 50% of maximum binding) calculated to 0.125 ± 0.022 µM and 0.183 ± 0.007 µM, respectively (Figure 3B). The sigmoid curve plotted in Figure 3B represents the EMSA data in Figure 3A. The binding experiments were, however, repeated several times and consistently revealed higher affinity for AP:G than for AP:A.Figure 3


Uracil-DNA glycosylases SMUG1 and UNG2 coordinate the initial steps of base excision repair by distinct mechanisms.

Pettersen HS, Sundheim O, Gilljam KM, Slupphaug G, Krokan HE, Kavli B - Nucleic Acids Res. (2007)

Product binding, effects of the AP-endonuclease APE1 and effects on AP-endonucleases APE1 and Exo III. (A) EMSA analysis of SMUG1 and UNG2 on AP-sites; 50, 100, 200, 300, 400 and 500 nM hSMUG1 and hUNG2 were incubated with 4 nM ds oligonucleotide (with complementary bases as indicated) or ss oligonucleotide, following uracil-excision binding to the product AP-sites were analysed by non-denaturating PAGE. (B) Percent bound AP:G and AP:A oligo plotted against the concentration of SMUG1 (µM) using a sigmoid curve fit model in GraphPad Prism®. The Kd values were calculated and represent the concentration of SMUG1 giving 50% of maximal binding to the AP-oligo. (C) Multiple turnover oligonucleotide assay with 0.85 nM SMUG1 and 20 nM U:G substrate (solid lines) or 20 nM Uss substrate (dotted lines) in the absence or presence of 8.5 nM APE1. (D) Multiple turnover oligonucleotide assay with 0.085 nM UNG2 and 20 nM U:G substrate (solid lines) or 20 nM Uss substrate (dotted lines) in the absence or presence of 8.5 nM APE1. Uracil excision was quantified after piperidine cleavage of the AP-site and separation by PAGE. (E) AP-cleavage activity of APE1 and ExoIII in the presence of SMUG1. (F) AP-cleavage activity of APE1 and ExoIII in the presence of UNG2. In (E) and (F), the AP-endonucleases (0.025 nM) were incubated for 10 min with 2 nM oligonucleotide substrate containing a central AP-site opposite guanine (AP:G) together with increasing amounts (0–1 µM) of SMUG1 or UNG2. AP-endonucleolytic cleavage was quantified after separation by PAGE.
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Figure 3: Product binding, effects of the AP-endonuclease APE1 and effects on AP-endonucleases APE1 and Exo III. (A) EMSA analysis of SMUG1 and UNG2 on AP-sites; 50, 100, 200, 300, 400 and 500 nM hSMUG1 and hUNG2 were incubated with 4 nM ds oligonucleotide (with complementary bases as indicated) or ss oligonucleotide, following uracil-excision binding to the product AP-sites were analysed by non-denaturating PAGE. (B) Percent bound AP:G and AP:A oligo plotted against the concentration of SMUG1 (µM) using a sigmoid curve fit model in GraphPad Prism®. The Kd values were calculated and represent the concentration of SMUG1 giving 50% of maximal binding to the AP-oligo. (C) Multiple turnover oligonucleotide assay with 0.85 nM SMUG1 and 20 nM U:G substrate (solid lines) or 20 nM Uss substrate (dotted lines) in the absence or presence of 8.5 nM APE1. (D) Multiple turnover oligonucleotide assay with 0.085 nM UNG2 and 20 nM U:G substrate (solid lines) or 20 nM Uss substrate (dotted lines) in the absence or presence of 8.5 nM APE1. Uracil excision was quantified after piperidine cleavage of the AP-site and separation by PAGE. (E) AP-cleavage activity of APE1 and ExoIII in the presence of SMUG1. (F) AP-cleavage activity of APE1 and ExoIII in the presence of UNG2. In (E) and (F), the AP-endonucleases (0.025 nM) were incubated for 10 min with 2 nM oligonucleotide substrate containing a central AP-site opposite guanine (AP:G) together with increasing amounts (0–1 µM) of SMUG1 or UNG2. AP-endonucleolytic cleavage was quantified after separation by PAGE.
Mentions: To elucidate the molecular mechanisms underlying the observed in vivo effects of SMUG1 and UNG2, we analysed the product (AP-site) binding subsequent to uracil-excision by purified human SMUG1 and UNG2 using electrophoretic mobility shift assays (EMSA). The results, illustrated in Figure 3A, demonstrate that SMUG1 readily binds to AP-sites in dsDNA (AP:G and AP:A), while no binding to AP-sites in single-stranded DNA (APss) or dsDNA without AP-site (T:A) was detected. In contrast, we did not observe binding of UNG2 to the same set of oligonucleotides. SMUG1 binds AP:G with slightly higher affinity than AP:A with Kd values (concentration of enzyme yielding 50% of maximum binding) calculated to 0.125 ± 0.022 µM and 0.183 ± 0.007 µM, respectively (Figure 3B). The sigmoid curve plotted in Figure 3B represents the EMSA data in Figure 3A. The binding experiments were, however, repeated several times and consistently revealed higher affinity for AP:G than for AP:A.Figure 3

Bottom Line: Mutations in this motif increase catalytic turnover due to reduced product binding.In contrast, the highly efficient UNG2 lacks product-binding capacity and stimulates AP-site cleavage by APE1, facilitating the two first steps in BER.In summary, this work reveals that SMUG1 and UNG2 coordinate the initial steps of BER by distinct mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Research and Molecular Medicine, NTNU, N-7006 Trondheim, Norway.

ABSTRACT
DNA glycosylases UNG and SMUG1 excise uracil from DNA and belong to the same protein superfamily. Vertebrates contain both SMUG1 and UNG, but their distinct roles in base excision repair (BER) of deaminated cytosine (U:G) are still not fully defined. Here we have examined the ability of human SMUG1 and UNG2 (nuclear UNG) to initiate and coordinate repair of U:G mismatches. When expressed in Escherichia coli cells, human UNG2 initiates complete repair of deaminated cytosine, while SMUG1 inhibits cell proliferation. In vitro, we show that SMUG1 binds tightly to AP-sites and inhibits AP-site cleavage by AP-endonucleases. Furthermore, a specific motif important for the AP-site product binding has been identified. Mutations in this motif increase catalytic turnover due to reduced product binding. In contrast, the highly efficient UNG2 lacks product-binding capacity and stimulates AP-site cleavage by APE1, facilitating the two first steps in BER. In summary, this work reveals that SMUG1 and UNG2 coordinate the initial steps of BER by distinct mechanisms. UNG2 is apparently adapted to rapid and highly coordinated repair of uracil (U:G and U:A) in replicating DNA, while the less efficient SMUG1 may be more important in repair of deaminated cytosine (U:G) in non-replicating chromatin.

Show MeSH
Related in: MedlinePlus