The formation of double-strand breaks at multiply damaged sites is driven by the kinetics of excision/incision at base damage in eukaryotic cells.
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.
Affiliation: CNRS UMR2027, Grenoble, France.
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.
Related in: MedlinePlus
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.