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The formation of double-strand breaks at multiply damaged sites is driven by the kinetics of excision/incision at base damage in eukaryotic cells.

Kozmin SG, Sedletska Y, Reynaud-Angelin A, Gasparutto D, Sage E - Nucleic Acids Res. (2009)

Bottom Line: In marked contrast, none of the MDS carrying opposed oG and hU separated by 3-8 bp gave rise to DSB, despite the fact that some of them contained preexisting single-strand break (a 1-nt gap).We propose that the kinetics of the initial repair steps at MDS is a major parameter that direct towards the conversion of MDS into DSB.Data provides clues to the biological consequences of MDS in eukaryotic cells.

View Article: PubMed Central - PubMed

Affiliation: CNRS UMR2027, Grenoble, France.

ABSTRACT
It has been stipulated that repair of clustered DNA lesions may be compromised, possibly leading to the formation of double-strand breaks (DSB) and, thus, to deleterious events. Using a variety of model multiply damaged sites (MDS), we investigated parameters that govern the formation of DSB during the processing of MDS. Duplexes carrying MDS were inserted into replicative or integrative vectors, and used to transform yeast Saccharomyces cerevisiae. Formation of DSB was assessed by a relevant plasmid survival assay. Kinetics of excision/incision and DSB formation at MDS was explored using yeast cell extracts. We show that MDS composed of two uracils or abasic sites, were rapidly incised and readily converted into DSB in yeast cells. In marked contrast, none of the MDS carrying opposed oG and hU separated by 3-8 bp gave rise to DSB, despite the fact that some of them contained preexisting single-strand break (a 1-nt gap). Interestingly, the absence of DSB formation in this case correlated with slow excision/incision rates of lesions. We propose that the kinetics of the initial repair steps at MDS is a major parameter that direct towards the conversion of MDS into DSB. Data provides clues to the biological consequences of MDS in eukaryotic cells.

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RTE of wild-type and repair deficient cells by replicative vector carrying diverse damaged duplex. Wild-type cells or ung1, apn1 apn2 or rad52 cells were transformed with pRS415 plasmid linearized by cleavage with SpeI and XhoI restriction enzymes (pRS415 XS, control ligation), or carrying SDS-oG, MDS-1, MDS-2, IMDS-oG4/hU, IMDS-oG7/hU, IMDS-oG9/hU, U/U, AP/AP, AP/U, U/AP constructs or undamaged oligonucleotide. RTE was calculated as the ratio of the transformation efficiency with pRS415 XS or with vector carrying a damaged duplex to the transformation efficiency with vector carrying undamaged oligonucleotide. Error bars represent the SD for at least three independent experiments. For each strain, in each independent experiment, the same stock of competent cells was transformed with all the tested MDS.
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Figure 2: RTE of wild-type and repair deficient cells by replicative vector carrying diverse damaged duplex. Wild-type cells or ung1, apn1 apn2 or rad52 cells were transformed with pRS415 plasmid linearized by cleavage with SpeI and XhoI restriction enzymes (pRS415 XS, control ligation), or carrying SDS-oG, MDS-1, MDS-2, IMDS-oG4/hU, IMDS-oG7/hU, IMDS-oG9/hU, U/U, AP/AP, AP/U, U/AP constructs or undamaged oligonucleotide. RTE was calculated as the ratio of the transformation efficiency with pRS415 XS or with vector carrying a damaged duplex to the transformation efficiency with vector carrying undamaged oligonucleotide. Error bars represent the SD for at least three independent experiments. For each strain, in each independent experiment, the same stock of competent cells was transformed with all the tested MDS.

Mentions: Wild-type yeast strain FF18733 and its isogenic derivatives defective in BER (ung1 ntg1 ntg2 ogg1 mag1 lacking all yeast DNA glycosylases of BER, and ntg1 ntg2), in NER (rad14), in both BER and NER (rad14 ntg1 ntg2) and in translesion DNA synthesis (rev3) were transformed with centromeric plasmid pRS415 carrying either MDS-1-containing oligonucleotide or undamaged oligonucleotide (Supplementary Figure S1a). Since the transformation efficiency by linear vector is very low, we assumed that the transformation efficiencies with vectors carrying the MDS would reflect the extent of DSB formed as repair intermediate. Surprisingly, we found that the transformation efficiencies with MDS-1-containing plasmid were similar to that with plasmid carrying undamaged oligonucleotide, in all tested strains (Table 1). This result may indicate that the repair process of MDS-1 did not generate a DSB that could lead to plasmid loss. Moreover, normal plasmid survival in rad51 or rad52 mutants (Table 1 and Figure 2) suggests that the major pathway of DSB repair in yeast, homologous recombination, is not involved in plasmid maintenance.Figure 2.


The formation of double-strand breaks at multiply damaged sites is driven by the kinetics of excision/incision at base damage in eukaryotic cells.

Kozmin SG, Sedletska Y, Reynaud-Angelin A, Gasparutto D, Sage E - Nucleic Acids Res. (2009)

RTE of wild-type and repair deficient cells by replicative vector carrying diverse damaged duplex. Wild-type cells or ung1, apn1 apn2 or rad52 cells were transformed with pRS415 plasmid linearized by cleavage with SpeI and XhoI restriction enzymes (pRS415 XS, control ligation), or carrying SDS-oG, MDS-1, MDS-2, IMDS-oG4/hU, IMDS-oG7/hU, IMDS-oG9/hU, U/U, AP/AP, AP/U, U/AP constructs or undamaged oligonucleotide. RTE was calculated as the ratio of the transformation efficiency with pRS415 XS or with vector carrying a damaged duplex to the transformation efficiency with vector carrying undamaged oligonucleotide. Error bars represent the SD for at least three independent experiments. For each strain, in each independent experiment, the same stock of competent cells was transformed with all the tested MDS.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 2: RTE of wild-type and repair deficient cells by replicative vector carrying diverse damaged duplex. Wild-type cells or ung1, apn1 apn2 or rad52 cells were transformed with pRS415 plasmid linearized by cleavage with SpeI and XhoI restriction enzymes (pRS415 XS, control ligation), or carrying SDS-oG, MDS-1, MDS-2, IMDS-oG4/hU, IMDS-oG7/hU, IMDS-oG9/hU, U/U, AP/AP, AP/U, U/AP constructs or undamaged oligonucleotide. RTE was calculated as the ratio of the transformation efficiency with pRS415 XS or with vector carrying a damaged duplex to the transformation efficiency with vector carrying undamaged oligonucleotide. Error bars represent the SD for at least three independent experiments. For each strain, in each independent experiment, the same stock of competent cells was transformed with all the tested MDS.
Mentions: Wild-type yeast strain FF18733 and its isogenic derivatives defective in BER (ung1 ntg1 ntg2 ogg1 mag1 lacking all yeast DNA glycosylases of BER, and ntg1 ntg2), in NER (rad14), in both BER and NER (rad14 ntg1 ntg2) and in translesion DNA synthesis (rev3) were transformed with centromeric plasmid pRS415 carrying either MDS-1-containing oligonucleotide or undamaged oligonucleotide (Supplementary Figure S1a). Since the transformation efficiency by linear vector is very low, we assumed that the transformation efficiencies with vectors carrying the MDS would reflect the extent of DSB formed as repair intermediate. Surprisingly, we found that the transformation efficiencies with MDS-1-containing plasmid were similar to that with plasmid carrying undamaged oligonucleotide, in all tested strains (Table 1). This result may indicate that the repair process of MDS-1 did not generate a DSB that could lead to plasmid loss. Moreover, normal plasmid survival in rad51 or rad52 mutants (Table 1 and Figure 2) suggests that the major pathway of DSB repair in yeast, homologous recombination, is not involved in plasmid maintenance.Figure 2.

Bottom Line: In marked contrast, none of the MDS carrying opposed oG and hU separated by 3-8 bp gave rise to DSB, despite the fact that some of them contained preexisting single-strand break (a 1-nt gap).We propose that the kinetics of the initial repair steps at MDS is a major parameter that direct towards the conversion of MDS into DSB.Data provides clues to the biological consequences of MDS in eukaryotic cells.

View Article: PubMed Central - PubMed

Affiliation: CNRS UMR2027, Grenoble, France.

ABSTRACT
It has been stipulated that repair of clustered DNA lesions may be compromised, possibly leading to the formation of double-strand breaks (DSB) and, thus, to deleterious events. Using a variety of model multiply damaged sites (MDS), we investigated parameters that govern the formation of DSB during the processing of MDS. Duplexes carrying MDS were inserted into replicative or integrative vectors, and used to transform yeast Saccharomyces cerevisiae. Formation of DSB was assessed by a relevant plasmid survival assay. Kinetics of excision/incision and DSB formation at MDS was explored using yeast cell extracts. We show that MDS composed of two uracils or abasic sites, were rapidly incised and readily converted into DSB in yeast cells. In marked contrast, none of the MDS carrying opposed oG and hU separated by 3-8 bp gave rise to DSB, despite the fact that some of them contained preexisting single-strand break (a 1-nt gap). Interestingly, the absence of DSB formation in this case correlated with slow excision/incision rates of lesions. We propose that the kinetics of the initial repair steps at MDS is a major parameter that direct towards the conversion of MDS into DSB. Data provides clues to the biological consequences of MDS in eukaryotic cells.

Show MeSH
Related in: MedlinePlus