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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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Repair of deaminated cytosine; model illustrating distinct coordination of BER initiated by SMUG1 and UNG2 in non-replicating chromatin and in replicating chromatin (foci), respectively. (A) 1. SMUG1 binds to the lesion and interacts with both strands in the DNA-helix. Uracil is probably flipped out of the helix and into the active site. 2. The catalysis is not very efficient because the active site is relaxed to be able to bind several other lesions. SMUG1 stays bound to the AP-site after excision. 3. APE1 competes with SMUG1 for AP-site binding. 4. SMUG1 is released form the product and is free to bind new lesions. 5. APE1 cuts the DNA strand, and Polβ/XRCC1/LigIIIα is recruited and completes BER. (B) 1. UNG2 is likely part of a highly coordinated and efficient repair complex scanning for lesions (U:G) in front of the replication fork (UNG2 is localized in replication foci). 2. Encountering the lesion uracil is flipped out of the DNA-helix and into the highly specific catalytic pocket of UNG2. Uracil is released by efficient hydrolysis of the N-glycosidic bond. Note, UNG2 interacts only with the uracil-containing DNA strand. UNG2 is immediately released from the AP-site and APE1 binds. 3. UNG2 stimulates APE1 cleavage of the AP-site and BER is completed.
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Figure 8: Repair of deaminated cytosine; model illustrating distinct coordination of BER initiated by SMUG1 and UNG2 in non-replicating chromatin and in replicating chromatin (foci), respectively. (A) 1. SMUG1 binds to the lesion and interacts with both strands in the DNA-helix. Uracil is probably flipped out of the helix and into the active site. 2. The catalysis is not very efficient because the active site is relaxed to be able to bind several other lesions. SMUG1 stays bound to the AP-site after excision. 3. APE1 competes with SMUG1 for AP-site binding. 4. SMUG1 is released form the product and is free to bind new lesions. 5. APE1 cuts the DNA strand, and Polβ/XRCC1/LigIIIα is recruited and completes BER. (B) 1. UNG2 is likely part of a highly coordinated and efficient repair complex scanning for lesions (U:G) in front of the replication fork (UNG2 is localized in replication foci). 2. Encountering the lesion uracil is flipped out of the DNA-helix and into the highly specific catalytic pocket of UNG2. Uracil is released by efficient hydrolysis of the N-glycosidic bond. Note, UNG2 interacts only with the uracil-containing DNA strand. UNG2 is immediately released from the AP-site and APE1 binds. 3. UNG2 stimulates APE1 cleavage of the AP-site and BER is completed.

Mentions: Taken together, it is clear that SMUG1 and UNG2 have evolved distinct mechanisms for the coordination of the second step in BER. Based on previous results and the new data presented here, we propose a model for how SMUG1 and UNG2 initiates and coordinates repair of deaminated cytosine (U:G) by distinct ‘hand-over’ mechanisms (Figure 8). This model is consistent with a role for SMUG1 in repair of deaminated cytosine in non-replicating chromatin and repair of uracil (U:G and U:A) by UNG2 in replication foci. The catalytically highly efficient and context-independent UNG2 enzyme is probably important in rapidly dividing cells to remove deaminated cytosine in front of the moving replication fork (pre-replicative repair), in addition to post-replicative repair of misincorporated uracil (13). This pre-replicative repair of U:G by UNG2 is supported by the observed 5.2-fold increased mutation frequency in Ung-deficient mouse embryonic fibroblasts (MEFs), mostly G:C to A:T transitions (8). SMUG1, on the other hand, is not designed to rapidly repair uracil during replication, and is probably more important in non-replicating chromatin, outside S-phase and in resting cells where the level of UNG2 is low (15). However, SMUG1 counteracts mutations also in cycling mouse cells (MEFs). Knocking down Smug1 by siRNA in MEFs resulted in 2.4-fold increased mutation frequency at the HPRT locus (8). Thus, the slow-acting, product-binding SMUG1 may efficiently recognize deaminated and some oxidized cytosine derivatives in non-replicating dsDNA (especially in A-T rich regions where the cytosine deamination rate is expected to be higher due to increased DNA breathing), excise the lesion and remain attached to the cytotoxic AP-site product until APE1 arrives and initiates further repair.Figure 8.


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)

Repair of deaminated cytosine; model illustrating distinct coordination of BER initiated by SMUG1 and UNG2 in non-replicating chromatin and in replicating chromatin (foci), respectively. (A) 1. SMUG1 binds to the lesion and interacts with both strands in the DNA-helix. Uracil is probably flipped out of the helix and into the active site. 2. The catalysis is not very efficient because the active site is relaxed to be able to bind several other lesions. SMUG1 stays bound to the AP-site after excision. 3. APE1 competes with SMUG1 for AP-site binding. 4. SMUG1 is released form the product and is free to bind new lesions. 5. APE1 cuts the DNA strand, and Polβ/XRCC1/LigIIIα is recruited and completes BER. (B) 1. UNG2 is likely part of a highly coordinated and efficient repair complex scanning for lesions (U:G) in front of the replication fork (UNG2 is localized in replication foci). 2. Encountering the lesion uracil is flipped out of the DNA-helix and into the highly specific catalytic pocket of UNG2. Uracil is released by efficient hydrolysis of the N-glycosidic bond. Note, UNG2 interacts only with the uracil-containing DNA strand. UNG2 is immediately released from the AP-site and APE1 binds. 3. UNG2 stimulates APE1 cleavage of the AP-site and BER is completed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 8: Repair of deaminated cytosine; model illustrating distinct coordination of BER initiated by SMUG1 and UNG2 in non-replicating chromatin and in replicating chromatin (foci), respectively. (A) 1. SMUG1 binds to the lesion and interacts with both strands in the DNA-helix. Uracil is probably flipped out of the helix and into the active site. 2. The catalysis is not very efficient because the active site is relaxed to be able to bind several other lesions. SMUG1 stays bound to the AP-site after excision. 3. APE1 competes with SMUG1 for AP-site binding. 4. SMUG1 is released form the product and is free to bind new lesions. 5. APE1 cuts the DNA strand, and Polβ/XRCC1/LigIIIα is recruited and completes BER. (B) 1. UNG2 is likely part of a highly coordinated and efficient repair complex scanning for lesions (U:G) in front of the replication fork (UNG2 is localized in replication foci). 2. Encountering the lesion uracil is flipped out of the DNA-helix and into the highly specific catalytic pocket of UNG2. Uracil is released by efficient hydrolysis of the N-glycosidic bond. Note, UNG2 interacts only with the uracil-containing DNA strand. UNG2 is immediately released from the AP-site and APE1 binds. 3. UNG2 stimulates APE1 cleavage of the AP-site and BER is completed.
Mentions: Taken together, it is clear that SMUG1 and UNG2 have evolved distinct mechanisms for the coordination of the second step in BER. Based on previous results and the new data presented here, we propose a model for how SMUG1 and UNG2 initiates and coordinates repair of deaminated cytosine (U:G) by distinct ‘hand-over’ mechanisms (Figure 8). This model is consistent with a role for SMUG1 in repair of deaminated cytosine in non-replicating chromatin and repair of uracil (U:G and U:A) by UNG2 in replication foci. The catalytically highly efficient and context-independent UNG2 enzyme is probably important in rapidly dividing cells to remove deaminated cytosine in front of the moving replication fork (pre-replicative repair), in addition to post-replicative repair of misincorporated uracil (13). This pre-replicative repair of U:G by UNG2 is supported by the observed 5.2-fold increased mutation frequency in Ung-deficient mouse embryonic fibroblasts (MEFs), mostly G:C to A:T transitions (8). SMUG1, on the other hand, is not designed to rapidly repair uracil during replication, and is probably more important in non-replicating chromatin, outside S-phase and in resting cells where the level of UNG2 is low (15). However, SMUG1 counteracts mutations also in cycling mouse cells (MEFs). Knocking down Smug1 by siRNA in MEFs resulted in 2.4-fold increased mutation frequency at the HPRT locus (8). Thus, the slow-acting, product-binding SMUG1 may efficiently recognize deaminated and some oxidized cytosine derivatives in non-replicating dsDNA (especially in A-T rich regions where the cytosine deamination rate is expected to be higher due to increased DNA breathing), excise the lesion and remain attached to the cytotoxic AP-site product until APE1 arrives and initiates further repair.Figure 8.

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