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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 but not UNG distinguishes U•G and U•A in duplex DNAs. (A) Electrophoretic mobility shift assay of purified hMutSα (16, 32, 65 or 130 nM) binding to labeled DNA duplexes containing U•G mispairs (left) or U•A pairs (right). Arrows indicate bound and free DNA. The percentage of DNA bound is shown below. (B) Quantitation of binding of purified MutSα to radiolabeled U•G duplexes in the presence of indicated levels of unlabeled U•G or U•A duplex competitor. (C) Products of deglycosylation of DNA duplex substrates containing U•G mispairs or U•A pairs by 0, 0.2, 0.4, 0.8, or 1.6 nM of purified hUNG, resolved by denaturing gel electrophoresis. Substrates were 5' end labeled on the uracil-containing strand, and following incubation with hUNG were treated with alkali to hydrolyze the backbone at abasic sites. The fraction of cleaved molecules, quantified by phosphorimager, is shown below each lane.
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Figure 1: MutSα but not UNG distinguishes U•G and U•A in duplex DNAs. (A) Electrophoretic mobility shift assay of purified hMutSα (16, 32, 65 or 130 nM) binding to labeled DNA duplexes containing U•G mispairs (left) or U•A pairs (right). Arrows indicate bound and free DNA. The percentage of DNA bound is shown below. (B) Quantitation of binding of purified MutSα to radiolabeled U•G duplexes in the presence of indicated levels of unlabeled U•G or U•A duplex competitor. (C) Products of deglycosylation of DNA duplex substrates containing U•G mispairs or U•A pairs by 0, 0.2, 0.4, 0.8, or 1.6 nM of purified hUNG, resolved by denaturing gel electrophoresis. Substrates were 5' end labeled on the uracil-containing strand, and following incubation with hUNG were treated with alkali to hydrolyze the backbone at abasic sites. The fraction of cleaved molecules, quantified by phosphorimager, is shown below each lane.

Mentions: We compared the binding of human MutSα (hMutSα) to synthetic DNA substrates containing U opposite either G (U•G) or A (U•A), using a gel mobility shift assay. hMutSα bound well to substrates containing U•G mismatches (apparent kD of 70 nM; Figure 1A), but poorly to substrates containing U•A pairs, (apparent kD > 135 nM). Competition analysis showed that binding of hMutSα to a labeled duplex substrate containing a single U•G mismatch was unaffected by the presence of a 50-fold molar excess of unlabeled duplex containing a U•A pair, but diminished more than 5-fold by a comparable amount of duplex DNA containing a U•G mismatch (Figure 1B). These properties are consistent with previous binding studies showing that hMutSα preferentially recognizes U•G or UU/GG heteroduplex oligonucleotides relative to homoduplexes [15-17]; and evidence that duplexes containing U•G but not U•A pairs activate MutSα ATPase activity [18].


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

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

MutSα but not UNG distinguishes U•G and U•A in duplex DNAs. (A) Electrophoretic mobility shift assay of purified hMutSα (16, 32, 65 or 130 nM) binding to labeled DNA duplexes containing U•G mispairs (left) or U•A pairs (right). Arrows indicate bound and free DNA. The percentage of DNA bound is shown below. (B) Quantitation of binding of purified MutSα to radiolabeled U•G duplexes in the presence of indicated levels of unlabeled U•G or U•A duplex competitor. (C) Products of deglycosylation of DNA duplex substrates containing U•G mispairs or U•A pairs by 0, 0.2, 0.4, 0.8, or 1.6 nM of purified hUNG, resolved by denaturing gel electrophoresis. Substrates were 5' end labeled on the uracil-containing strand, and following incubation with hUNG were treated with alkali to hydrolyze the backbone at abasic sites. The fraction of cleaved molecules, quantified by phosphorimager, is shown below each lane.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: MutSα but not UNG distinguishes U•G and U•A in duplex DNAs. (A) Electrophoretic mobility shift assay of purified hMutSα (16, 32, 65 or 130 nM) binding to labeled DNA duplexes containing U•G mispairs (left) or U•A pairs (right). Arrows indicate bound and free DNA. The percentage of DNA bound is shown below. (B) Quantitation of binding of purified MutSα to radiolabeled U•G duplexes in the presence of indicated levels of unlabeled U•G or U•A duplex competitor. (C) Products of deglycosylation of DNA duplex substrates containing U•G mispairs or U•A pairs by 0, 0.2, 0.4, 0.8, or 1.6 nM of purified hUNG, resolved by denaturing gel electrophoresis. Substrates were 5' end labeled on the uracil-containing strand, and following incubation with hUNG were treated with alkali to hydrolyze the backbone at abasic sites. The fraction of cleaved molecules, quantified by phosphorimager, is shown below each lane.
Mentions: We compared the binding of human MutSα (hMutSα) to synthetic DNA substrates containing U opposite either G (U•G) or A (U•A), using a gel mobility shift assay. hMutSα bound well to substrates containing U•G mismatches (apparent kD of 70 nM; Figure 1A), but poorly to substrates containing U•A pairs, (apparent kD > 135 nM). Competition analysis showed that binding of hMutSα to a labeled duplex substrate containing a single U•G mismatch was unaffected by the presence of a 50-fold molar excess of unlabeled duplex containing a U•A pair, but diminished more than 5-fold by a comparable amount of duplex DNA containing a U•G mismatch (Figure 1B). These properties are consistent with previous binding studies showing that hMutSα preferentially recognizes U•G or UU/GG heteroduplex oligonucleotides relative to homoduplexes [15-17]; and evidence that duplexes containing U•G but not U•A pairs activate MutSα ATPase activity [18].

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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