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The Smc5/6 complex is required for dissolution of DNA-mediated sister chromatid linkages.

Bermúdez-López M, Ceschia A, de Piccoli G, Colomina N, Pasero P, Aragón L, Torres-Rosell J - Nucleic Acids Res. (2010)

Bottom Line: Here, we show that the dissolution of these connections is an active process that requires the Smc5/6 complex, together with Mms21, its associated SUMO-ligase.Failure to remove DNA-mediated linkages causes gross chromosome missegregation in anaphase.Moreover, we show that Smc5/6 is capable to dissolve them in metaphase-arrested cells, thus restoring chromosome resolution and segregation.

View Article: PubMed Central - PubMed

Affiliation: IRBLLEIDA, Department of Ciències Mèdiques Bàsiques, Facultat de Medicina, Universitat de Lleida, 25008 Lleida, Spain.

ABSTRACT
Mitotic chromosome segregation requires the removal of physical connections between sister chromatids. In addition to cohesin and topological entrapments, sister chromatid separation can be prevented by the presence of chromosome junctions or ongoing DNA replication. We will collectively refer to them as DNA-mediated linkages. Although this type of structures has been documented in different DNA replication and repair mutants, there is no known essential mechanism ensuring their timely removal before mitosis. Here, we show that the dissolution of these connections is an active process that requires the Smc5/6 complex, together with Mms21, its associated SUMO-ligase. Failure to remove DNA-mediated linkages causes gross chromosome missegregation in anaphase. Moreover, we show that Smc5/6 is capable to dissolve them in metaphase-arrested cells, thus restoring chromosome resolution and segregation. We propose that Smc5/6 has an essential role in the removal of DNA-mediated linkages to prevent chromosome missegregation and aneuploidy.

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Smc6 mutant cells accumulate SCJs and unfinished replication intermediates after MMS damage. Wild-type and smc6-9 mutant cells were treated as in Figure 2D. Cells were subsequently blocked in metaphase with nocodazole. (A) Samples were taken for 2D gel analysis and probed for the same region studied in Figure 1. Note the presence of replication intermediates and SCJs in smc6-9 cells. (B) Cells were treated as in (A) except that they were released from the G1 block in the presence of BrdU to label replicated DNA. Once arrested in G2/M, samples were taken for DNA combing. Absolute values for number of examined fibers and total DNA examined; mean values for length of fibers, gaps, BrdU tracks and gaps per megabase. (C) Representative fibers from wild-type and smc6-9 cells. Red, DNA; green, BrdU; white, BrdU channel alone; asterisk, unreplicated gaps. Bar, 50 kb.
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Figure 3: Smc6 mutant cells accumulate SCJs and unfinished replication intermediates after MMS damage. Wild-type and smc6-9 mutant cells were treated as in Figure 2D. Cells were subsequently blocked in metaphase with nocodazole. (A) Samples were taken for 2D gel analysis and probed for the same region studied in Figure 1. Note the presence of replication intermediates and SCJs in smc6-9 cells. (B) Cells were treated as in (A) except that they were released from the G1 block in the presence of BrdU to label replicated DNA. Once arrested in G2/M, samples were taken for DNA combing. Absolute values for number of examined fibers and total DNA examined; mean values for length of fibers, gaps, BrdU tracks and gaps per megabase. (C) Representative fibers from wild-type and smc6-9 cells. Red, DNA; green, BrdU; white, BrdU channel alone; asterisk, unreplicated gaps. Bar, 50 kb.

Mentions: We next studied the nature of the linkages that were preventing chromosome segregation in smc6 mutants. The segregation defects indicated that these structures should still be present at the time of anaphase onset. We therefore treated G1 arrested cells with a pulse of MMS and studied their DNA after G1 release into a metaphase block. Under these conditions, the levels of replication and recombination intermediates are almost undetectable in wild-type cells by 2D gel electrophoresis (Figure 3A). In contrast, smc6-9 mutant cells display low but readily detectable levels of X-shaped DNA and replication forks in the ARS305 region on chromosome 3. This result indicates that the MMS pulse conditions used in this study are inducing the accumulation of SCJs in smc6-9 cells. Chromosome linkages can also be generated by the presence of replication forks. In fact, smc6-9 mutant cells accumulate both unfinished replication and SCJs in the rDNA array at the time of chromosome segregation (10,19). Therefore, we checked replication of individual chromosomes in smc6 cells by DNA combing. Wild-type and smc6-9 mutant cells were treated with a pulse of MMS in G1. Cells were then released into S phase in the presence of BrdU to label replicated DNA. Nocodazole was added to arrest cells in metaphase. Intact chromosomal DNA was prepared and DNA fibers were treated as described (25). The number of unreplicated gaps (BrdU-negative) was scored and the overall percentage of DNA replication was calculated on the basis of the number and length of gaps. It should be noted that the gap frequency in wild-type cells is not affected by the MMS pulse; besides, unchallenged smc6-9 cells display the same frequency of replication gaps as wild-type cells (data not shown). In contrast, smc6-9 mutant cells display ∼2.5 times increase in the number of unreplicated gaps relative to wild-type cells after a G1 pulse of MMS (Figure 3B and C). The estimated failure in DNA replication completion increases from 6.5% in wild-type cells to 16.3% in smc6-9 mutant cells. Additionally, the average length of chromosome fibers is significantly shorter in smc6-9 cells, suggesting that smc6-9 fibers are more fragile. This could be due to the presence of sister chromatid linkages, which could also minimize the calculated amount of unreplicated chromosomal DNA. It is worth noting that, although the percentages of unreplicated gaps are relatively high in both strains, most of the chromosomes have been replicated in the metaphase arrest. We conclude that the Smc5/6 complex is required for completion of genome replication in response to a pulse of alkylation DNA damage. Therefore, the persistent chromosomal links in smc6 cells arises from, at least, two different types of structures, i.e. incomplete replication and X-shaped DNA structures.Figure 3.


The Smc5/6 complex is required for dissolution of DNA-mediated sister chromatid linkages.

Bermúdez-López M, Ceschia A, de Piccoli G, Colomina N, Pasero P, Aragón L, Torres-Rosell J - Nucleic Acids Res. (2010)

Smc6 mutant cells accumulate SCJs and unfinished replication intermediates after MMS damage. Wild-type and smc6-9 mutant cells were treated as in Figure 2D. Cells were subsequently blocked in metaphase with nocodazole. (A) Samples were taken for 2D gel analysis and probed for the same region studied in Figure 1. Note the presence of replication intermediates and SCJs in smc6-9 cells. (B) Cells were treated as in (A) except that they were released from the G1 block in the presence of BrdU to label replicated DNA. Once arrested in G2/M, samples were taken for DNA combing. Absolute values for number of examined fibers and total DNA examined; mean values for length of fibers, gaps, BrdU tracks and gaps per megabase. (C) Representative fibers from wild-type and smc6-9 cells. Red, DNA; green, BrdU; white, BrdU channel alone; asterisk, unreplicated gaps. Bar, 50 kb.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: Smc6 mutant cells accumulate SCJs and unfinished replication intermediates after MMS damage. Wild-type and smc6-9 mutant cells were treated as in Figure 2D. Cells were subsequently blocked in metaphase with nocodazole. (A) Samples were taken for 2D gel analysis and probed for the same region studied in Figure 1. Note the presence of replication intermediates and SCJs in smc6-9 cells. (B) Cells were treated as in (A) except that they were released from the G1 block in the presence of BrdU to label replicated DNA. Once arrested in G2/M, samples were taken for DNA combing. Absolute values for number of examined fibers and total DNA examined; mean values for length of fibers, gaps, BrdU tracks and gaps per megabase. (C) Representative fibers from wild-type and smc6-9 cells. Red, DNA; green, BrdU; white, BrdU channel alone; asterisk, unreplicated gaps. Bar, 50 kb.
Mentions: We next studied the nature of the linkages that were preventing chromosome segregation in smc6 mutants. The segregation defects indicated that these structures should still be present at the time of anaphase onset. We therefore treated G1 arrested cells with a pulse of MMS and studied their DNA after G1 release into a metaphase block. Under these conditions, the levels of replication and recombination intermediates are almost undetectable in wild-type cells by 2D gel electrophoresis (Figure 3A). In contrast, smc6-9 mutant cells display low but readily detectable levels of X-shaped DNA and replication forks in the ARS305 region on chromosome 3. This result indicates that the MMS pulse conditions used in this study are inducing the accumulation of SCJs in smc6-9 cells. Chromosome linkages can also be generated by the presence of replication forks. In fact, smc6-9 mutant cells accumulate both unfinished replication and SCJs in the rDNA array at the time of chromosome segregation (10,19). Therefore, we checked replication of individual chromosomes in smc6 cells by DNA combing. Wild-type and smc6-9 mutant cells were treated with a pulse of MMS in G1. Cells were then released into S phase in the presence of BrdU to label replicated DNA. Nocodazole was added to arrest cells in metaphase. Intact chromosomal DNA was prepared and DNA fibers were treated as described (25). The number of unreplicated gaps (BrdU-negative) was scored and the overall percentage of DNA replication was calculated on the basis of the number and length of gaps. It should be noted that the gap frequency in wild-type cells is not affected by the MMS pulse; besides, unchallenged smc6-9 cells display the same frequency of replication gaps as wild-type cells (data not shown). In contrast, smc6-9 mutant cells display ∼2.5 times increase in the number of unreplicated gaps relative to wild-type cells after a G1 pulse of MMS (Figure 3B and C). The estimated failure in DNA replication completion increases from 6.5% in wild-type cells to 16.3% in smc6-9 mutant cells. Additionally, the average length of chromosome fibers is significantly shorter in smc6-9 cells, suggesting that smc6-9 fibers are more fragile. This could be due to the presence of sister chromatid linkages, which could also minimize the calculated amount of unreplicated chromosomal DNA. It is worth noting that, although the percentages of unreplicated gaps are relatively high in both strains, most of the chromosomes have been replicated in the metaphase arrest. We conclude that the Smc5/6 complex is required for completion of genome replication in response to a pulse of alkylation DNA damage. Therefore, the persistent chromosomal links in smc6 cells arises from, at least, two different types of structures, i.e. incomplete replication and X-shaped DNA structures.Figure 3.

Bottom Line: Here, we show that the dissolution of these connections is an active process that requires the Smc5/6 complex, together with Mms21, its associated SUMO-ligase.Failure to remove DNA-mediated linkages causes gross chromosome missegregation in anaphase.Moreover, we show that Smc5/6 is capable to dissolve them in metaphase-arrested cells, thus restoring chromosome resolution and segregation.

View Article: PubMed Central - PubMed

Affiliation: IRBLLEIDA, Department of Ciències Mèdiques Bàsiques, Facultat de Medicina, Universitat de Lleida, 25008 Lleida, Spain.

ABSTRACT
Mitotic chromosome segregation requires the removal of physical connections between sister chromatids. In addition to cohesin and topological entrapments, sister chromatid separation can be prevented by the presence of chromosome junctions or ongoing DNA replication. We will collectively refer to them as DNA-mediated linkages. Although this type of structures has been documented in different DNA replication and repair mutants, there is no known essential mechanism ensuring their timely removal before mitosis. Here, we show that the dissolution of these connections is an active process that requires the Smc5/6 complex, together with Mms21, its associated SUMO-ligase. Failure to remove DNA-mediated linkages causes gross chromosome missegregation in anaphase. Moreover, we show that Smc5/6 is capable to dissolve them in metaphase-arrested cells, thus restoring chromosome resolution and segregation. We propose that Smc5/6 has an essential role in the removal of DNA-mediated linkages to prevent chromosome missegregation and aneuploidy.

Show MeSH
Related in: MedlinePlus