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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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Related in: MedlinePlus

Duplexes carrying damaged sites used in this study. Oligonucleotides carried 8-oxoguanine (oG), 8-oxoadenine (oA), 5-hydroxyuracil (hU), 5-formyluracil (fU), uracil (U) or abasic site (AP) (obtained by conversion of uracil to AP site by UDG). Duplexes containing a U and an AP site in the two orientations were also used. oG was located at 4 bp from hU in MDS-1, MDS-2 and IMDS-oG4/hU, at 7 and 9 bp from hU in IMDS-oG7/hU and IMDS-oG9/hU, respectively. MDS-1 and MDS-2 carried a 1-nt gap terminated by 3′-OH and 5′-OH. Undamaged (upper) and damaged duplexes are 56-bp long and harbored SpeI and XhoI restriction sites at each extremity.
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Figure 1: Duplexes carrying damaged sites used in this study. Oligonucleotides carried 8-oxoguanine (oG), 8-oxoadenine (oA), 5-hydroxyuracil (hU), 5-formyluracil (fU), uracil (U) or abasic site (AP) (obtained by conversion of uracil to AP site by UDG). Duplexes containing a U and an AP site in the two orientations were also used. oG was located at 4 bp from hU in MDS-1, MDS-2 and IMDS-oG4/hU, at 7 and 9 bp from hU in IMDS-oG7/hU and IMDS-oG9/hU, respectively. MDS-1 and MDS-2 carried a 1-nt gap terminated by 3′-OH and 5′-OH. Undamaged (upper) and damaged duplexes are 56-bp long and harbored SpeI and XhoI restriction sites at each extremity.

Mentions: Unmodified oligonucleotides and oligonucleotides carrying uracil were purchased from Sigma-Proligo (France). Oligonucleotides carrying 8-oxoguanine (oG), 8-oxoadenine (oA), 5-hydroxyuracil (hU) and 5-formyluracil (fU) were synthesized as previously described (18). With these oligonucleotides, we built various MDS constructs of different orientation and complexities, as depicted in Figure 1. Duplexes carrying abasic (AP) sites were obtained by hybridization of single-stranded uracil-containing oligonucleotides digested with UDG protein (see below preparation of radiolabeled oligonucleotides). All duplexes harbor SpeI and XhoI restriction sites (Figure 1). Low-copy centromeric plasmid pRS415 (Stratagene) and integrative vector YIplac204-LPG (constructed as described in Supplementary Material; see Supplementary Figure S1a for plasmid maps) were used for introduction of oligonucleotides into yeast.Figure 1.


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)

Duplexes carrying damaged sites used in this study. Oligonucleotides carried 8-oxoguanine (oG), 8-oxoadenine (oA), 5-hydroxyuracil (hU), 5-formyluracil (fU), uracil (U) or abasic site (AP) (obtained by conversion of uracil to AP site by UDG). Duplexes containing a U and an AP site in the two orientations were also used. oG was located at 4 bp from hU in MDS-1, MDS-2 and IMDS-oG4/hU, at 7 and 9 bp from hU in IMDS-oG7/hU and IMDS-oG9/hU, respectively. MDS-1 and MDS-2 carried a 1-nt gap terminated by 3′-OH and 5′-OH. Undamaged (upper) and damaged duplexes are 56-bp long and harbored SpeI and XhoI restriction sites at each extremity.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665211&req=5

Figure 1: Duplexes carrying damaged sites used in this study. Oligonucleotides carried 8-oxoguanine (oG), 8-oxoadenine (oA), 5-hydroxyuracil (hU), 5-formyluracil (fU), uracil (U) or abasic site (AP) (obtained by conversion of uracil to AP site by UDG). Duplexes containing a U and an AP site in the two orientations were also used. oG was located at 4 bp from hU in MDS-1, MDS-2 and IMDS-oG4/hU, at 7 and 9 bp from hU in IMDS-oG7/hU and IMDS-oG9/hU, respectively. MDS-1 and MDS-2 carried a 1-nt gap terminated by 3′-OH and 5′-OH. Undamaged (upper) and damaged duplexes are 56-bp long and harbored SpeI and XhoI restriction sites at each extremity.
Mentions: Unmodified oligonucleotides and oligonucleotides carrying uracil were purchased from Sigma-Proligo (France). Oligonucleotides carrying 8-oxoguanine (oG), 8-oxoadenine (oA), 5-hydroxyuracil (hU) and 5-formyluracil (fU) were synthesized as previously described (18). With these oligonucleotides, we built various MDS constructs of different orientation and complexities, as depicted in Figure 1. Duplexes carrying abasic (AP) sites were obtained by hybridization of single-stranded uracil-containing oligonucleotides digested with UDG protein (see below preparation of radiolabeled oligonucleotides). All duplexes harbor SpeI and XhoI restriction sites (Figure 1). Low-copy centromeric plasmid pRS415 (Stratagene) and integrative vector YIplac204-LPG (constructed as described in Supplementary Material; see Supplementary Figure S1a for plasmid maps) were used for introduction of oligonucleotides into yeast.Figure 1.

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