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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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The model proposed for processing of MDS composed by two oxidized bases and U. We propose that U-oG/hU MDS are processed by three pathways. A major pathway (∼80%) converts MDS into DSB in WT cells owing to the excision/incision at U and hU by Ung, EndoIII (Nth), AP endonucleases, whereas it does not occur in Δung cells. This was determined from the fraction of viable colonies (∼20%) after transformation with MDS-containing DNA. For ∼19% of the events in WT cells, the fast excision/incision at U produces an SSB, which, in the presence of surrounding lesions, is not further repaired and leads to the loss of the U-oG-strand. Error-prone replication of hU-carrying strand thus occurs and leads to elevated mutagenesis at hU (reaching almost 100% of surviving clones). This pathway is not activated in Δung strain. In rare cases (∼1% of total events in WT cell), when the full repair of U is extremely fast (WT) or does not occur (in Δung), the hU is repaired before oG and the excision/incision at hU partly inhibits or retards repair of oG. Consequently, the mutagenesis rate at hU within MDS is as at single hU (in WT cells), and mutagenesis at oG within studied MDS is slightly increased in comparison with that at single oG. This third pathway represents the only possible pathway in Δung cells. It should be slightly more represented in the processing of MDS/−5 than for MDS/+1 because of greater interlesion spacing.
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gkt731-F6: The model proposed for processing of MDS composed by two oxidized bases and U. We propose that U-oG/hU MDS are processed by three pathways. A major pathway (∼80%) converts MDS into DSB in WT cells owing to the excision/incision at U and hU by Ung, EndoIII (Nth), AP endonucleases, whereas it does not occur in Δung cells. This was determined from the fraction of viable colonies (∼20%) after transformation with MDS-containing DNA. For ∼19% of the events in WT cells, the fast excision/incision at U produces an SSB, which, in the presence of surrounding lesions, is not further repaired and leads to the loss of the U-oG-strand. Error-prone replication of hU-carrying strand thus occurs and leads to elevated mutagenesis at hU (reaching almost 100% of surviving clones). This pathway is not activated in Δung strain. In rare cases (∼1% of total events in WT cell), when the full repair of U is extremely fast (WT) or does not occur (in Δung), the hU is repaired before oG and the excision/incision at hU partly inhibits or retards repair of oG. Consequently, the mutagenesis rate at hU within MDS is as at single hU (in WT cells), and mutagenesis at oG within studied MDS is slightly increased in comparison with that at single oG. This third pathway represents the only possible pathway in Δung cells. It should be slightly more represented in the processing of MDS/−5 than for MDS/+1 because of greater interlesion spacing.

Mentions: Such data lead us to propose a model for the processing of MDS composed of oxidized purines and pyrimidines and one U or AP site and in which U and an oxidized pyrimidine are on opposite strands (Figure 6). According to this model, in WT cells, the majority of MDS is converted to deleterious DSB. In this pathway, the activities of the AP endonuclease following that of Ung and that of EndoIII (or oxidized pyrimidine DNA n-glycosylases) on their cognate lesions trigger DSB formation. The presence of two Us/AP sites on opposite strands would increase the extent of DSB. Conversely, this pathway is inhibited in cells lacking DNA n-glycosylases or AP endonucleases. However, in WT cells a small fraction of MDS escapes conversion to the DSB, and the processing of lesions within MDS leads to strand loss. As most DNA n-glycosylases are unable to excise base damage on single-stranded DNA, this pathway is a major actor in mutagenesis. Nevertheless, the mutagenesis rate will depend on the capacity of the lesion left unrepaired on the remaining strand to be bypassed in error-free or error-prone manner. It may range from 90% for hU to no mutation for DHT, or to ∼4% of mutagenesis at oG on single-stranded DNA (57). Finally, a small fraction of MDS (<1%) is processed without DSB formation or mutagenesis induction. This implies sequential repair of each of the comprised lesions. In this last process, the interlesion spacing should play a role. However, in a strain deficient for Ung, the inductions of DSB and of strand loss do not occur, unveiling a third pathway potentially involved in the processing of such MDS.Figure 6.


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)

The model proposed for processing of MDS composed by two oxidized bases and U. We propose that U-oG/hU MDS are processed by three pathways. A major pathway (∼80%) converts MDS into DSB in WT cells owing to the excision/incision at U and hU by Ung, EndoIII (Nth), AP endonucleases, whereas it does not occur in Δung cells. This was determined from the fraction of viable colonies (∼20%) after transformation with MDS-containing DNA. For ∼19% of the events in WT cells, the fast excision/incision at U produces an SSB, which, in the presence of surrounding lesions, is not further repaired and leads to the loss of the U-oG-strand. Error-prone replication of hU-carrying strand thus occurs and leads to elevated mutagenesis at hU (reaching almost 100% of surviving clones). This pathway is not activated in Δung strain. In rare cases (∼1% of total events in WT cell), when the full repair of U is extremely fast (WT) or does not occur (in Δung), the hU is repaired before oG and the excision/incision at hU partly inhibits or retards repair of oG. Consequently, the mutagenesis rate at hU within MDS is as at single hU (in WT cells), and mutagenesis at oG within studied MDS is slightly increased in comparison with that at single oG. This third pathway represents the only possible pathway in Δung cells. It should be slightly more represented in the processing of MDS/−5 than for MDS/+1 because of greater interlesion spacing.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt731-F6: The model proposed for processing of MDS composed by two oxidized bases and U. We propose that U-oG/hU MDS are processed by three pathways. A major pathway (∼80%) converts MDS into DSB in WT cells owing to the excision/incision at U and hU by Ung, EndoIII (Nth), AP endonucleases, whereas it does not occur in Δung cells. This was determined from the fraction of viable colonies (∼20%) after transformation with MDS-containing DNA. For ∼19% of the events in WT cells, the fast excision/incision at U produces an SSB, which, in the presence of surrounding lesions, is not further repaired and leads to the loss of the U-oG-strand. Error-prone replication of hU-carrying strand thus occurs and leads to elevated mutagenesis at hU (reaching almost 100% of surviving clones). This pathway is not activated in Δung strain. In rare cases (∼1% of total events in WT cell), when the full repair of U is extremely fast (WT) or does not occur (in Δung), the hU is repaired before oG and the excision/incision at hU partly inhibits or retards repair of oG. Consequently, the mutagenesis rate at hU within MDS is as at single hU (in WT cells), and mutagenesis at oG within studied MDS is slightly increased in comparison with that at single oG. This third pathway represents the only possible pathway in Δung cells. It should be slightly more represented in the processing of MDS/−5 than for MDS/+1 because of greater interlesion spacing.
Mentions: Such data lead us to propose a model for the processing of MDS composed of oxidized purines and pyrimidines and one U or AP site and in which U and an oxidized pyrimidine are on opposite strands (Figure 6). According to this model, in WT cells, the majority of MDS is converted to deleterious DSB. In this pathway, the activities of the AP endonuclease following that of Ung and that of EndoIII (or oxidized pyrimidine DNA n-glycosylases) on their cognate lesions trigger DSB formation. The presence of two Us/AP sites on opposite strands would increase the extent of DSB. Conversely, this pathway is inhibited in cells lacking DNA n-glycosylases or AP endonucleases. However, in WT cells a small fraction of MDS escapes conversion to the DSB, and the processing of lesions within MDS leads to strand loss. As most DNA n-glycosylases are unable to excise base damage on single-stranded DNA, this pathway is a major actor in mutagenesis. Nevertheless, the mutagenesis rate will depend on the capacity of the lesion left unrepaired on the remaining strand to be bypassed in error-free or error-prone manner. It may range from 90% for hU to no mutation for DHT, or to ∼4% of mutagenesis at oG on single-stranded DNA (57). Finally, a small fraction of MDS (<1%) is processed without DSB formation or mutagenesis induction. This implies sequential repair of each of the comprised lesions. In this last process, the interlesion spacing should play a role. However, in a strain deficient for Ung, the inductions of DSB and of strand loss do not occur, unveiling a third pathway potentially involved in the processing of such MDS.Figure 6.

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