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High-fidelity correction of genomic uracil by human mismatch repair activities.

Larson ED, Bednarski DW, Maizels N - BMC Mol. Biol. (2008)

Bottom Line: Deamination of cytosine to produce uracil is a common and potentially mutagenic lesion in genomic DNA.U*G mismatches are also the initiating lesion in immunoglobulin gene diversification, where they undergo mutagenic processing by redundant pathways, one dependent upon uracil excision and the other upon mismatch recognition by MutS alpha.This contrasts with UNG, which readily excises U opposite either A or G.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, University of Washington School of Medicine, Seattle, WA 98195-7650, USA. elarson@ilstu.edu

ABSTRACT

Background: Deamination of cytosine to produce uracil is a common and potentially mutagenic lesion in genomic DNA. U*G mismatches occur spontaneously throughout the genome, where they are repaired by factors associated with the base excision repair pathway. U*G mismatches are also the initiating lesion in immunoglobulin gene diversification, where they undergo mutagenic processing by redundant pathways, one dependent upon uracil excision and the other upon mismatch recognition by MutS alpha. While UNG is well known to initiate repair of uracil in DNA, the ability of MutS alpha to direct correction of this base has not been directly demonstrated.

Results: Using a biochemical assay for mismatch repair, we show that MutS alpha can promote efficient and faithful repair of U*G mismatches, but does not repair U*A pairs in DNA. This contrasts with UNG, which readily excises U opposite either A or G. Repair of U*G by MutS alpha depends upon DNA polymerase delta (pol delta), ATP, and proliferating cell nuclear antigen (PCNA), all properties of canonical mismatch repair.

Conclusion: These results show that faithful repair of U*G can be carried out by either the mismatch repair or base excision repair pathways. Thus, the redundant functions of these pathways in immunoglobulin gene diversification reflect their redundant functions in faithful repair. Faithful repair by either pathway is comparably efficient, suggesting that mismatch repair and base excision repair share the task of faithful repair of genomic uracil.

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MutSα directs faithful repair of U•G mispairs in human nuclear extracts. (A) Representative reactions demonstrating repair of M13-U•G substrates by Ugi-treated LoVo nuclear extracts, in the absence (-) or presence (+) of 53 nM purified hMutSα. Arrows at left indicate unrepaired and repaired products; fraction of molecules repaired is shown below each lane. (B) Products of extension by pol η in the absence or presence of aphidicolin. Arrows at left indicate primer and extension product. (C) Representative reactions demonstrating repair of M13-U•G substrates by Ugi-treated HeLa or Ramos nuclear extracts, in the presence and absence of aphidicolin, which inhibits pol δ; PBP, the PCNA binding peptide, which inhibits PCNA; or exogenous ATP. Notations as in panel A.
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Figure 4: MutSα directs faithful repair of U•G mispairs in human nuclear extracts. (A) Representative reactions demonstrating repair of M13-U•G substrates by Ugi-treated LoVo nuclear extracts, in the absence (-) or presence (+) of 53 nM purified hMutSα. Arrows at left indicate unrepaired and repaired products; fraction of molecules repaired is shown below each lane. (B) Products of extension by pol η in the absence or presence of aphidicolin. Arrows at left indicate primer and extension product. (C) Representative reactions demonstrating repair of M13-U•G substrates by Ugi-treated HeLa or Ramos nuclear extracts, in the presence and absence of aphidicolin, which inhibits pol δ; PBP, the PCNA binding peptide, which inhibits PCNA; or exogenous ATP. Notations as in panel A.

Mentions: To ask if MutSα can direct repair of U•G mispairs, we assayed repair of the M13-U•G circular substrates in Ugi-treated extracts derived from the MSH2-deficient cell line LoVo. Mismatch repair is defective in LoVo extracts but can be restored in vitro by addition of purified hMutSα [23,24,32,36,37]. Nuclear extracts of LoVo, treated with Ugi to inhibit UNG (e.g. Figure 3A), supported repair of only 8% of the M13-U•G substrates (Figure 4A). Addition of purified hMutSα to Ugi-treated LoVo nuclear extracts increased repair 3-fold, to 24% (Figure 4A). Thus, MutSα promotes efficient repair of U•G mispairs.


High-fidelity correction of genomic uracil by human mismatch repair activities.

Larson ED, Bednarski DW, Maizels N - BMC Mol. Biol. (2008)

MutSα directs faithful repair of U•G mispairs in human nuclear extracts. (A) Representative reactions demonstrating repair of M13-U•G substrates by Ugi-treated LoVo nuclear extracts, in the absence (-) or presence (+) of 53 nM purified hMutSα. Arrows at left indicate unrepaired and repaired products; fraction of molecules repaired is shown below each lane. (B) Products of extension by pol η in the absence or presence of aphidicolin. Arrows at left indicate primer and extension product. (C) Representative reactions demonstrating repair of M13-U•G substrates by Ugi-treated HeLa or Ramos nuclear extracts, in the presence and absence of aphidicolin, which inhibits pol δ; PBP, the PCNA binding peptide, which inhibits PCNA; or exogenous ATP. Notations as in panel A.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2606688&req=5

Figure 4: MutSα directs faithful repair of U•G mispairs in human nuclear extracts. (A) Representative reactions demonstrating repair of M13-U•G substrates by Ugi-treated LoVo nuclear extracts, in the absence (-) or presence (+) of 53 nM purified hMutSα. Arrows at left indicate unrepaired and repaired products; fraction of molecules repaired is shown below each lane. (B) Products of extension by pol η in the absence or presence of aphidicolin. Arrows at left indicate primer and extension product. (C) Representative reactions demonstrating repair of M13-U•G substrates by Ugi-treated HeLa or Ramos nuclear extracts, in the presence and absence of aphidicolin, which inhibits pol δ; PBP, the PCNA binding peptide, which inhibits PCNA; or exogenous ATP. Notations as in panel A.
Mentions: To ask if MutSα can direct repair of U•G mispairs, we assayed repair of the M13-U•G circular substrates in Ugi-treated extracts derived from the MSH2-deficient cell line LoVo. Mismatch repair is defective in LoVo extracts but can be restored in vitro by addition of purified hMutSα [23,24,32,36,37]. Nuclear extracts of LoVo, treated with Ugi to inhibit UNG (e.g. Figure 3A), supported repair of only 8% of the M13-U•G substrates (Figure 4A). Addition of purified hMutSα to Ugi-treated LoVo nuclear extracts increased repair 3-fold, to 24% (Figure 4A). Thus, MutSα promotes efficient repair of U•G mispairs.

Bottom Line: Deamination of cytosine to produce uracil is a common and potentially mutagenic lesion in genomic DNA.U*G mismatches are also the initiating lesion in immunoglobulin gene diversification, where they undergo mutagenic processing by redundant pathways, one dependent upon uracil excision and the other upon mismatch recognition by MutS alpha.This contrasts with UNG, which readily excises U opposite either A or G.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, University of Washington School of Medicine, Seattle, WA 98195-7650, USA. elarson@ilstu.edu

ABSTRACT

Background: Deamination of cytosine to produce uracil is a common and potentially mutagenic lesion in genomic DNA. U*G mismatches occur spontaneously throughout the genome, where they are repaired by factors associated with the base excision repair pathway. U*G mismatches are also the initiating lesion in immunoglobulin gene diversification, where they undergo mutagenic processing by redundant pathways, one dependent upon uracil excision and the other upon mismatch recognition by MutS alpha. While UNG is well known to initiate repair of uracil in DNA, the ability of MutS alpha to direct correction of this base has not been directly demonstrated.

Results: Using a biochemical assay for mismatch repair, we show that MutS alpha can promote efficient and faithful repair of U*G mismatches, but does not repair U*A pairs in DNA. This contrasts with UNG, which readily excises U opposite either A or G. Repair of U*G by MutS alpha depends upon DNA polymerase delta (pol delta), ATP, and proliferating cell nuclear antigen (PCNA), all properties of canonical mismatch repair.

Conclusion: These results show that faithful repair of U*G can be carried out by either the mismatch repair or base excision repair pathways. Thus, the redundant functions of these pathways in immunoglobulin gene diversification reflect their redundant functions in faithful repair. Faithful repair by either pathway is comparably efficient, suggesting that mismatch repair and base excision repair share the task of faithful repair of genomic uracil.

Show MeSH
Related in: MedlinePlus