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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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Uracil-substrate preference of SMUG1 active-site mutants. Limited turnover oligonucleotide assay of SMUG1 mutants. Equimolar amounts (20 nM) of enzyme and [33P]-labelled uracil oligonucleotide substrate (U:G, U:A and Uss) were incubated for 5, 30 and 60 min. Uracil excision was quantified after piperidine cleavage of the AP-site and separation by denaturing PAGE.
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Figure 5: Uracil-substrate preference of SMUG1 active-site mutants. Limited turnover oligonucleotide assay of SMUG1 mutants. Equimolar amounts (20 nM) of enzyme and [33P]-labelled uracil oligonucleotide substrate (U:G, U:A and Uss) were incubated for 5, 30 and 60 min. Uracil excision was quantified after piperidine cleavage of the AP-site and separation by denaturing PAGE.

Mentions: To quantify residual activity of the hSMUG1 mutants towards U:G, we used a multiple turnover oligonucletide assay (see “Materials and Methods” section) with a 50-fold molar excess of U:G substrate. Interestingly, the residual activities of the mutants were even higher when analyzed with U:G substrate as compared with U:A substrate (Table 1). To minimize the effect of product binding, the SMUG1 mutants were also analysed using equimolar amounts of enzyme and oligonucleotides with uracil in either U:G, U:A or Uss contexts. Under these conditions, the U:G preference of the SMUG1 mutants was even more pronounced (Figure 5). A stable enzyme-substrate complex with a long residence time will increase the possibility for catalysis to occur, thus these results indicate that SMUG1 binds U:G substrate (not only AP:G product) with higher affinity than U:A and Uss substrates.Figure 5.


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)

Uracil-substrate preference of SMUG1 active-site mutants. Limited turnover oligonucleotide assay of SMUG1 mutants. Equimolar amounts (20 nM) of enzyme and [33P]-labelled uracil oligonucleotide substrate (U:G, U:A and Uss) were incubated for 5, 30 and 60 min. Uracil excision was quantified after piperidine cleavage of the AP-site and separation by denaturing PAGE.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919486&req=5

Figure 5: Uracil-substrate preference of SMUG1 active-site mutants. Limited turnover oligonucleotide assay of SMUG1 mutants. Equimolar amounts (20 nM) of enzyme and [33P]-labelled uracil oligonucleotide substrate (U:G, U:A and Uss) were incubated for 5, 30 and 60 min. Uracil excision was quantified after piperidine cleavage of the AP-site and separation by denaturing PAGE.
Mentions: To quantify residual activity of the hSMUG1 mutants towards U:G, we used a multiple turnover oligonucletide assay (see “Materials and Methods” section) with a 50-fold molar excess of U:G substrate. Interestingly, the residual activities of the mutants were even higher when analyzed with U:G substrate as compared with U:A substrate (Table 1). To minimize the effect of product binding, the SMUG1 mutants were also analysed using equimolar amounts of enzyme and oligonucleotides with uracil in either U:G, U:A or Uss contexts. Under these conditions, the U:G preference of the SMUG1 mutants was even more pronounced (Figure 5). A stable enzyme-substrate complex with a long residence time will increase the possibility for catalysis to occur, thus these results indicate that SMUG1 binds U:G substrate (not only AP:G product) with higher affinity than U:A and Uss substrates.Figure 5.

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