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Replication fork collapse is a major cause of the high mutation frequency at three-base lesion clusters.

Sedletska Y, Radicella JP, Sage E - Nucleic Acids Res. (2013)

Bottom Line: We designed multiply damaged sites (MDS) composed of a rapidly excised uracil (U) and two oxidized bases, 5-hydroxyuracil (hU) and 8-oxoguanine (oG), excised more slowly.Induction of DSB was estimated from plasmid survival and mutagenesis determined by sequencing of surviving clones.We show that a large majority of MDS is converted to DSB, whereas almost all surviving clones are mutated at hU.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, Centre de Recherche, F-91405 Orsay, France; CNRS UMR3348, F-91405 Orsay, France and CEA, Institut de Radiobiologie Cellulaire et Moléculaire, 18 route du Panorama, F-92265 Fontenay aux Roses, France.

ABSTRACT
Unresolved repair of clustered DNA lesions can lead to the formation of deleterious double strand breaks (DSB) or to mutation induction. Here, we investigated the outcome of clusters composed of base lesions for which base excision repair enzymes have different kinetics of excision/incision. We designed multiply damaged sites (MDS) composed of a rapidly excised uracil (U) and two oxidized bases, 5-hydroxyuracil (hU) and 8-oxoguanine (oG), excised more slowly. Plasmids harboring these U-oG/hU MDS-carrying duplexes were introduced into Escherichia coli cells either wild type or deficient for DNA n-glycosylases. Induction of DSB was estimated from plasmid survival and mutagenesis determined by sequencing of surviving clones. We show that a large majority of MDS is converted to DSB, whereas almost all surviving clones are mutated at hU. We demonstrate that mutagenesis at hU is correlated with excision of the U placed on the opposite strand. We propose that excision of U by Ung initiates the loss of U-oG-carrying strand, resulting in enhanced mutagenesis at the lesion present on the opposite strand. Our results highlight the importance of the kinetics of excision by base excision repair DNA n-glycosylases in the processing and fate of MDS and provide evidence for the role of strand loss/replication fork collapse during the processing of MDS on their mutational consequences.

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Cleavage efficiency at hU by human cell extracts. 32P-labeled duplexes SDS-hU, MDS/+1 or MDS/−5 were incubated with 30 µg of proteins from whole-cell extract at 37°C for various periods and separated on 12% denaturing polyacrylamide gels. Data represent the means of at least three independent experiments. Preparation of whole-cell extracts and analysis of cleavage activity were performed as described in (15).
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gkt731-F3: Cleavage efficiency at hU by human cell extracts. 32P-labeled duplexes SDS-hU, MDS/+1 or MDS/−5 were incubated with 30 µg of proteins from whole-cell extract at 37°C for various periods and separated on 12% denaturing polyacrylamide gels. Data represent the means of at least three independent experiments. Preparation of whole-cell extracts and analysis of cleavage activity were performed as described in (15).

Mentions: We examined the cleavage efficiency at hU and U in MDS/+1 and MDS/−5 using human whole-cell extract. Figure 3 shows that hU is efficiently cleaved, although at a slightly slower rate in comparison with single hU. The excision/incision at U in MDS/+1 and MDS/−5 is complete and fast (Supplementary Figure S3 in Supplementary Information) and, as expected, the rate of cleavage is higher than that at hU and similar to that of U/U by yeast extracts (26). Because the BER repair is well conserved from bacteria to mammalian cells, the high cleavage efficiency observed here at U and hU is consistent with the elevated induction of DSB at U-oG/hU in E. coli.Figure 3.


Replication fork collapse is a major cause of the high mutation frequency at three-base lesion clusters.

Sedletska Y, Radicella JP, Sage E - Nucleic Acids Res. (2013)

Cleavage efficiency at hU by human cell extracts. 32P-labeled duplexes SDS-hU, MDS/+1 or MDS/−5 were incubated with 30 µg of proteins from whole-cell extract at 37°C for various periods and separated on 12% denaturing polyacrylamide gels. Data represent the means of at least three independent experiments. Preparation of whole-cell extracts and analysis of cleavage activity were performed as described in (15).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814351&req=5

gkt731-F3: Cleavage efficiency at hU by human cell extracts. 32P-labeled duplexes SDS-hU, MDS/+1 or MDS/−5 were incubated with 30 µg of proteins from whole-cell extract at 37°C for various periods and separated on 12% denaturing polyacrylamide gels. Data represent the means of at least three independent experiments. Preparation of whole-cell extracts and analysis of cleavage activity were performed as described in (15).
Mentions: We examined the cleavage efficiency at hU and U in MDS/+1 and MDS/−5 using human whole-cell extract. Figure 3 shows that hU is efficiently cleaved, although at a slightly slower rate in comparison with single hU. The excision/incision at U in MDS/+1 and MDS/−5 is complete and fast (Supplementary Figure S3 in Supplementary Information) and, as expected, the rate of cleavage is higher than that at hU and similar to that of U/U by yeast extracts (26). Because the BER repair is well conserved from bacteria to mammalian cells, the high cleavage efficiency observed here at U and hU is consistent with the elevated induction of DSB at U-oG/hU in E. coli.Figure 3.

Bottom Line: We designed multiply damaged sites (MDS) composed of a rapidly excised uracil (U) and two oxidized bases, 5-hydroxyuracil (hU) and 8-oxoguanine (oG), excised more slowly.Induction of DSB was estimated from plasmid survival and mutagenesis determined by sequencing of surviving clones.We show that a large majority of MDS is converted to DSB, whereas almost all surviving clones are mutated at hU.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, Centre de Recherche, F-91405 Orsay, France; CNRS UMR3348, F-91405 Orsay, France and CEA, Institut de Radiobiologie Cellulaire et Moléculaire, 18 route du Panorama, F-92265 Fontenay aux Roses, France.

ABSTRACT
Unresolved repair of clustered DNA lesions can lead to the formation of deleterious double strand breaks (DSB) or to mutation induction. Here, we investigated the outcome of clusters composed of base lesions for which base excision repair enzymes have different kinetics of excision/incision. We designed multiply damaged sites (MDS) composed of a rapidly excised uracil (U) and two oxidized bases, 5-hydroxyuracil (hU) and 8-oxoguanine (oG), excised more slowly. Plasmids harboring these U-oG/hU MDS-carrying duplexes were introduced into Escherichia coli cells either wild type or deficient for DNA n-glycosylases. Induction of DSB was estimated from plasmid survival and mutagenesis determined by sequencing of surviving clones. We show that a large majority of MDS is converted to DSB, whereas almost all surviving clones are mutated at hU. We demonstrate that mutagenesis at hU is correlated with excision of the U placed on the opposite strand. We propose that excision of U by Ung initiates the loss of U-oG-carrying strand, resulting in enhanced mutagenesis at the lesion present on the opposite strand. Our results highlight the importance of the kinetics of excision by base excision repair DNA n-glycosylases in the processing and fate of MDS and provide evidence for the role of strand loss/replication fork collapse during the processing of MDS on their mutational consequences.

Show MeSH
Related in: MedlinePlus