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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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Cleavage efficiency at uracil, AP sites, oG and hU by yeast whole-cell extracts. 32P-labeled duplexes U/U, AP/AP or MDS-2 were incubated with 20 μg of proteins from whole-cell extracts at 37°C for various periods of time and separated on 12% denaturing polyacrylamide gels. Two different cell extracts were used. Data represent the means of at least two independent experiments for each extract. Kinetics of cleavage at U or at AP were similar on the two strands and similar to that in AP/U and U/AP. Kinetics of cleavage at oG and hU are given for MDS-2, but they are similar to that in IMDS-oG4/hU or SDS-oG and SDS-hU.
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Figure 3: Cleavage efficiency at uracil, AP sites, oG and hU by yeast whole-cell extracts. 32P-labeled duplexes U/U, AP/AP or MDS-2 were incubated with 20 μg of proteins from whole-cell extracts at 37°C for various periods of time and separated on 12% denaturing polyacrylamide gels. Two different cell extracts were used. Data represent the means of at least two independent experiments for each extract. Kinetics of cleavage at U or at AP were similar on the two strands and similar to that in AP/U and U/AP. Kinetics of cleavage at oG and hU are given for MDS-2, but they are similar to that in IMDS-oG4/hU or SDS-oG and SDS-hU.

Mentions: Using yeast cell-free extracts, we analyzed the rate of cleavage at uracil, AP site, hU and oG in our damaged duplex on denaturing gels and also examined the rate of DSB formation by native gel electrophoresis. Figure 3 shows the rate of cleavage at uracil, AP, hU and oG in duplexes U/U, AP/AP and MDS-2. The results demonstrate that uracil and AP site were extremely quickly excised, in comparison with hU or oG. As observed with mammalian cell extracts, initial rate of cleavage at hU is higher than at oG for all MDS tested (Figure 3 and data not shown). In addition, the rates of cleavage at oG or hU observed for IMDS-oG4/hU and MDS-1 were similar to those shown in Figure 3 for MDS-2 (data not shown). The extent of cleavage at hU and oG could indicate that incision may occur on different molecules. The induction of DSB was thus checked. We observed (Figure 4a) that within 5 min a large part of the initial duplex containing AP sites was converted into cleaved products, while after 4 h incubation with the extract about half of MDS-1 remained uncleaved. Figure 4b reveals that most of DSB were produced within a few minutes in all duplexes containing uracil and/or AP sites, and that after 30 min of incubation about 95% of DSB were formed in these initial duplexes, whereas only 35% and 10% of DSB were formed in MDS-2 and MDS-1, respectively. The plateau for DSB formation reached virtually 100% for duplexes containing uracil and/or AP sites, while a maximum of 50% and 60% of substrate was converted into DSB for MDS-1 and MDS-2, respectively, under our experimental conditions. Interestingly, cleavage at hU and oG in IMDS-oG4/hU occurred mainly on separated molecules, as revealed by the very low extent of DSB formed (10% after 4 h incubation). These observations imply that in MDS-1 and MDS-2, the formation of DSB is largely favored by the presence of the 1-nt gap which cannot be repaired under our in vitro experimental conditions (18). Altogether, these data demonstrate that when excision of a damage is very fast, like for uracil and AP sites, excision occurs simultaneously on both strands leading to the formation of DSB (as long as there is no steric hindrance for two proteins at the binding site). In contrast, a lower excision rate protects from the formation of DSB, which may be mainly induced in the presence of pre-existing SSB, at least in vitro.Figure 3.


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)

Cleavage efficiency at uracil, AP sites, oG and hU by yeast whole-cell extracts. 32P-labeled duplexes U/U, AP/AP or MDS-2 were incubated with 20 μg of proteins from whole-cell extracts at 37°C for various periods of time and separated on 12% denaturing polyacrylamide gels. Two different cell extracts were used. Data represent the means of at least two independent experiments for each extract. Kinetics of cleavage at U or at AP were similar on the two strands and similar to that in AP/U and U/AP. Kinetics of cleavage at oG and hU are given for MDS-2, but they are similar to that in IMDS-oG4/hU or SDS-oG and SDS-hU.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Cleavage efficiency at uracil, AP sites, oG and hU by yeast whole-cell extracts. 32P-labeled duplexes U/U, AP/AP or MDS-2 were incubated with 20 μg of proteins from whole-cell extracts at 37°C for various periods of time and separated on 12% denaturing polyacrylamide gels. Two different cell extracts were used. Data represent the means of at least two independent experiments for each extract. Kinetics of cleavage at U or at AP were similar on the two strands and similar to that in AP/U and U/AP. Kinetics of cleavage at oG and hU are given for MDS-2, but they are similar to that in IMDS-oG4/hU or SDS-oG and SDS-hU.
Mentions: Using yeast cell-free extracts, we analyzed the rate of cleavage at uracil, AP site, hU and oG in our damaged duplex on denaturing gels and also examined the rate of DSB formation by native gel electrophoresis. Figure 3 shows the rate of cleavage at uracil, AP, hU and oG in duplexes U/U, AP/AP and MDS-2. The results demonstrate that uracil and AP site were extremely quickly excised, in comparison with hU or oG. As observed with mammalian cell extracts, initial rate of cleavage at hU is higher than at oG for all MDS tested (Figure 3 and data not shown). In addition, the rates of cleavage at oG or hU observed for IMDS-oG4/hU and MDS-1 were similar to those shown in Figure 3 for MDS-2 (data not shown). The extent of cleavage at hU and oG could indicate that incision may occur on different molecules. The induction of DSB was thus checked. We observed (Figure 4a) that within 5 min a large part of the initial duplex containing AP sites was converted into cleaved products, while after 4 h incubation with the extract about half of MDS-1 remained uncleaved. Figure 4b reveals that most of DSB were produced within a few minutes in all duplexes containing uracil and/or AP sites, and that after 30 min of incubation about 95% of DSB were formed in these initial duplexes, whereas only 35% and 10% of DSB were formed in MDS-2 and MDS-1, respectively. The plateau for DSB formation reached virtually 100% for duplexes containing uracil and/or AP sites, while a maximum of 50% and 60% of substrate was converted into DSB for MDS-1 and MDS-2, respectively, under our experimental conditions. Interestingly, cleavage at hU and oG in IMDS-oG4/hU occurred mainly on separated molecules, as revealed by the very low extent of DSB formed (10% after 4 h incubation). These observations imply that in MDS-1 and MDS-2, the formation of DSB is largely favored by the presence of the 1-nt gap which cannot be repaired under our in vitro experimental conditions (18). Altogether, these data demonstrate that when excision of a damage is very fast, like for uracil and AP sites, excision occurs simultaneously on both strands leading to the formation of DSB (as long as there is no steric hindrance for two proteins at the binding site). In contrast, a lower excision rate protects from the formation of DSB, which may be mainly induced in the presence of pre-existing SSB, at least in vitro.Figure 3.

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