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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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Related in: MedlinePlus

2D gel electrophoresis of replication intermediates digested so that the (GAA)35 repeat was in the middle of the fragment. (A) The scheme of the digest of pUCneoGAA/CTT35 by BsaI and NcoI. (B) 2D gel electrophoresis of replication intermediates of pUCneo digested with BsaI and NcoI. A single bulge (combined spot 1, and spot 2, Figure 1B) was observed upon BsaI–NcoI digest. A position of the spot 3 is also shown. We concluded that replication approached the GAA repeat from both ends of the fragment.
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gks021-F3: 2D gel electrophoresis of replication intermediates digested so that the (GAA)35 repeat was in the middle of the fragment. (A) The scheme of the digest of pUCneoGAA/CTT35 by BsaI and NcoI. (B) 2D gel electrophoresis of replication intermediates of pUCneo digested with BsaI and NcoI. A single bulge (combined spot 1, and spot 2, Figure 1B) was observed upon BsaI–NcoI digest. A position of the spot 3 is also shown. We concluded that replication approached the GAA repeat from both ends of the fragment.

Mentions: To confirm this idea, we digested the same replication intermediates with the NcoI and BsaI restriction enzymes, positioning the GAA run in the center of the fragment (Figure 3A). In this case, forks entering the fragment from its opposite ends should both reach the repeat at the middle of the fragment, i.e. the two bulges (1 and 2) should now convene at the fragment's center (Figure 3A). This turned out to be the case: one can clearly see a unique bulge in the middle of the arc, with a single spike emanating from it. We believe that the spikes connecting spots 1 and 2 to spot 3 result from replication forks entering the fragment from both ends: one of the forks is stalled, and another approaches from the opposite direction (Figure 3B).Figure 3.


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)

2D gel electrophoresis of replication intermediates digested so that the (GAA)35 repeat was in the middle of the fragment. (A) The scheme of the digest of pUCneoGAA/CTT35 by BsaI and NcoI. (B) 2D gel electrophoresis of replication intermediates of pUCneo digested with BsaI and NcoI. A single bulge (combined spot 1, and spot 2, Figure 1B) was observed upon BsaI–NcoI digest. A position of the spot 3 is also shown. We concluded that replication approached the GAA repeat from both ends of the fragment.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks021-F3: 2D gel electrophoresis of replication intermediates digested so that the (GAA)35 repeat was in the middle of the fragment. (A) The scheme of the digest of pUCneoGAA/CTT35 by BsaI and NcoI. (B) 2D gel electrophoresis of replication intermediates of pUCneo digested with BsaI and NcoI. A single bulge (combined spot 1, and spot 2, Figure 1B) was observed upon BsaI–NcoI digest. A position of the spot 3 is also shown. We concluded that replication approached the GAA repeat from both ends of the fragment.
Mentions: To confirm this idea, we digested the same replication intermediates with the NcoI and BsaI restriction enzymes, positioning the GAA run in the center of the fragment (Figure 3A). In this case, forks entering the fragment from its opposite ends should both reach the repeat at the middle of the fragment, i.e. the two bulges (1 and 2) should now convene at the fragment's center (Figure 3A). This turned out to be the case: one can clearly see a unique bulge in the middle of the arc, with a single spike emanating from it. We believe that the spikes connecting spots 1 and 2 to spot 3 result from replication forks entering the fragment from both ends: one of the forks is stalled, and another approaches from the opposite direction (Figure 3B).Figure 3.

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