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Effects of Friedreich's ataxia GAA repeats on DNA replication in mammalian cells.

Chandok GS, Patel MP, Mirkin SM, Krasilnikova MM - Nucleic Acids Res. (2012)

Bottom Line: Here we studied the effects of (GAA)n repeats of varying lengths and orientations on the episomal DNA replication in mammalian cells.We have recently shown that the very first round of the transfected DNA replication occurs in the lack of the mature chromatin, does not depend on the episomal replication origin and initiates at multiple single-stranded regions of plasmid DNA.We now found that expanded GAA repeats severely block this first replication round post plasmid transfection, while the subsequent replication cycles are only mildly affected.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Penn State University, University Park, PA 16802, USA.

ABSTRACT
Friedreich's ataxia (FRDA) is a common hereditary degenerative neuro-muscular disorder caused by expansions of the (GAA)n repeat in the first intron of the frataxin gene. The expanded repeats from parents frequently undergo further significant length changes as they are passed on to progeny. Expanded repeats also show an age-dependent instability in somatic cells, albeit on a smaller scale than during intergenerational transmissions. Here we studied the effects of (GAA)n repeats of varying lengths and orientations on the episomal DNA replication in mammalian cells. We have recently shown that the very first round of the transfected DNA replication occurs in the lack of the mature chromatin, does not depend on the episomal replication origin and initiates at multiple single-stranded regions of plasmid DNA. We now found that expanded GAA repeats severely block this first replication round post plasmid transfection, while the subsequent replication cycles are only mildly affected. The fact that GAA repeats affect various replication modes in a different way might shed light on their differential expansions characteristic for FRDA.

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(A) The first round of replication of the plasmid containing GAA repeat is more pronounced than that of a control plasmid. The same amount of plasmids Ori(-)CTT57 and Ori(-) were used for 293A transfections. The cells were lysed 6 h after transfection, replication intermediates were isolated and digested with AflIII. The samples were intentionally run together to allow direct comparison. (B) The samples Ori(-)CTT57 and Ori(-) contain about the same amount of plasmid.
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gks021-F7: (A) The first round of replication of the plasmid containing GAA repeat is more pronounced than that of a control plasmid. The same amount of plasmids Ori(-)CTT57 and Ori(-) were used for 293A transfections. The cells were lysed 6 h after transfection, replication intermediates were isolated and digested with AflIII. The samples were intentionally run together to allow direct comparison. (B) The samples Ori(-)CTT57 and Ori(-) contain about the same amount of plasmid.

Mentions: As discussed above, the elongation stage of the alternative replication is severely inhibited by the presence of even relatively short GAA repeats. Remarkably, however, the same repeats seem to increase the rate of alternative replication initiation. This is evident, for example, from the fact that the intensity of the replication arc approaching the repeat is much higher than that for the control, repeat-free plasmid (Figure 7A). Note that about the same amount of the plasmid was loaded for GAA-containing and control plasmids (Figure 7B). Similar increases in the alternative replication efficiency of the repeat-containing plasmids were consistently observed in all our experiments, which required us to load significantly more replication intermediates for the control plasmid to equalize them with the intermediates of the repeat-containing plasmids (Figure 6B).Figure 7.


Effects of Friedreich's ataxia GAA repeats on DNA replication in mammalian cells.

Chandok GS, Patel MP, Mirkin SM, Krasilnikova MM - Nucleic Acids Res. (2012)

(A) The first round of replication of the plasmid containing GAA repeat is more pronounced than that of a control plasmid. The same amount of plasmids Ori(-)CTT57 and Ori(-) were used for 293A transfections. The cells were lysed 6 h after transfection, replication intermediates were isolated and digested with AflIII. The samples were intentionally run together to allow direct comparison. (B) The samples Ori(-)CTT57 and Ori(-) contain about the same amount of plasmid.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351192&req=5

gks021-F7: (A) The first round of replication of the plasmid containing GAA repeat is more pronounced than that of a control plasmid. The same amount of plasmids Ori(-)CTT57 and Ori(-) were used for 293A transfections. The cells were lysed 6 h after transfection, replication intermediates were isolated and digested with AflIII. The samples were intentionally run together to allow direct comparison. (B) The samples Ori(-)CTT57 and Ori(-) contain about the same amount of plasmid.
Mentions: As discussed above, the elongation stage of the alternative replication is severely inhibited by the presence of even relatively short GAA repeats. Remarkably, however, the same repeats seem to increase the rate of alternative replication initiation. This is evident, for example, from the fact that the intensity of the replication arc approaching the repeat is much higher than that for the control, repeat-free plasmid (Figure 7A). Note that about the same amount of the plasmid was loaded for GAA-containing and control plasmids (Figure 7B). Similar increases in the alternative replication efficiency of the repeat-containing plasmids were consistently observed in all our experiments, which required us to load significantly more replication intermediates for the control plasmid to equalize them with the intermediates of the repeat-containing plasmids (Figure 6B).Figure 7.

Bottom Line: Here we studied the effects of (GAA)n repeats of varying lengths and orientations on the episomal DNA replication in mammalian cells.We have recently shown that the very first round of the transfected DNA replication occurs in the lack of the mature chromatin, does not depend on the episomal replication origin and initiates at multiple single-stranded regions of plasmid DNA.We now found that expanded GAA repeats severely block this first replication round post plasmid transfection, while the subsequent replication cycles are only mildly affected.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Penn State University, University Park, PA 16802, USA.

ABSTRACT
Friedreich's ataxia (FRDA) is a common hereditary degenerative neuro-muscular disorder caused by expansions of the (GAA)n repeat in the first intron of the frataxin gene. The expanded repeats from parents frequently undergo further significant length changes as they are passed on to progeny. Expanded repeats also show an age-dependent instability in somatic cells, albeit on a smaller scale than during intergenerational transmissions. Here we studied the effects of (GAA)n repeats of varying lengths and orientations on the episomal DNA replication in mammalian cells. We have recently shown that the very first round of the transfected DNA replication occurs in the lack of the mature chromatin, does not depend on the episomal replication origin and initiates at multiple single-stranded regions of plasmid DNA. We now found that expanded GAA repeats severely block this first replication round post plasmid transfection, while the subsequent replication cycles are only mildly affected. The fact that GAA repeats affect various replication modes in a different way might shed light on their differential expansions characteristic for FRDA.

Show MeSH
Related in: MedlinePlus