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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) Replication stalling was no longer observed upon DpnI digest of replication intermediates. 2D gel electrophoresis of the replication intermediates of pUCneoGAA57 isolated from the COS-1 cells and digested with either AflIII alone, or AflIII and DpnI. To eliminate semimethylated products of the first replication cycle, the digest was performed with 20 units of DpnI, and 10 units of AflIII for about 2 h. Bubble arc can be observed on the overexposed panel of pUCneoGAA57 digested with AflIII (shown by an arrow). (B) An expanded (GAA)230 repeat did not cause replication stalling of the T-antigen-driven replication. 2D gel electrophoresis of the replication intermediates of pUCneoGAA/CTT230 isolated from COS-1 cells and digested with AflIII and DpnI. (C) Replication stalling at the GAA repeat decreased over time. 2D gel electrophoresis of replication intermediates of the pUCneoGAA57 plasmid isolated at 2, 12, 18, 24 and 30 h after COS-1 cells transfection. Only gel-purified monomer plasmid was used for transfection. Replication intermediates were digested with AflIII. The descending part of the Y arc increased, while the bulges and the double Y arc decreased with incubation time.
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gks021-F5: (A) Replication stalling was no longer observed upon DpnI digest of replication intermediates. 2D gel electrophoresis of the replication intermediates of pUCneoGAA57 isolated from the COS-1 cells and digested with either AflIII alone, or AflIII and DpnI. To eliminate semimethylated products of the first replication cycle, the digest was performed with 20 units of DpnI, and 10 units of AflIII for about 2 h. Bubble arc can be observed on the overexposed panel of pUCneoGAA57 digested with AflIII (shown by an arrow). (B) An expanded (GAA)230 repeat did not cause replication stalling of the T-antigen-driven replication. 2D gel electrophoresis of the replication intermediates of pUCneoGAA/CTT230 isolated from COS-1 cells and digested with AflIII and DpnI. (C) Replication stalling at the GAA repeat decreased over time. 2D gel electrophoresis of replication intermediates of the pUCneoGAA57 plasmid isolated at 2, 12, 18, 24 and 30 h after COS-1 cells transfection. Only gel-purified monomer plasmid was used for transfection. Replication intermediates were digested with AflIII. The descending part of the Y arc increased, while the bulges and the double Y arc decreased with incubation time.

Mentions: Our previous analysis of the alternative replication mode revealed that it is limited to the first replication cycle after episomal transfection into mammalian cells (26). We believe that the pattern of stalling at GAA repeat that we observed (Figures 2–5) was a superposition of the first replication cycle that initiated randomly throughout the sequence, and SV40 origin-initiated replication. Since the first cycle replication initiated everywhere, it mostly produced Y arc, and some of the bubble arc that we could only observe at overexposed pictures (Figure 5A, overexposed panel). During the first replication cycle, replication intermediates consist of the template DNA strands that came from Escherichia coli plasmid and nascent strands synthesized in mammalian cells. In subsequent replication cycles, both nascent and template strands are of mammalian origin. This allows one to distinguish between the first and the subsequent replication cycles using a DpnI restriction digest. This enzyme cleaves GATC sequences when they are methylated or hemimethylated by the bacterial Dam methylase. Thus, the DpnI digest eliminates the non-replicated episomal DNA, as well as the products of its first replication cycle in mammalian cells.Figure 5.


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) Replication stalling was no longer observed upon DpnI digest of replication intermediates. 2D gel electrophoresis of the replication intermediates of pUCneoGAA57 isolated from the COS-1 cells and digested with either AflIII alone, or AflIII and DpnI. To eliminate semimethylated products of the first replication cycle, the digest was performed with 20 units of DpnI, and 10 units of AflIII for about 2 h. Bubble arc can be observed on the overexposed panel of pUCneoGAA57 digested with AflIII (shown by an arrow). (B) An expanded (GAA)230 repeat did not cause replication stalling of the T-antigen-driven replication. 2D gel electrophoresis of the replication intermediates of pUCneoGAA/CTT230 isolated from COS-1 cells and digested with AflIII and DpnI. (C) Replication stalling at the GAA repeat decreased over time. 2D gel electrophoresis of replication intermediates of the pUCneoGAA57 plasmid isolated at 2, 12, 18, 24 and 30 h after COS-1 cells transfection. Only gel-purified monomer plasmid was used for transfection. Replication intermediates were digested with AflIII. The descending part of the Y arc increased, while the bulges and the double Y arc decreased with incubation time.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks021-F5: (A) Replication stalling was no longer observed upon DpnI digest of replication intermediates. 2D gel electrophoresis of the replication intermediates of pUCneoGAA57 isolated from the COS-1 cells and digested with either AflIII alone, or AflIII and DpnI. To eliminate semimethylated products of the first replication cycle, the digest was performed with 20 units of DpnI, and 10 units of AflIII for about 2 h. Bubble arc can be observed on the overexposed panel of pUCneoGAA57 digested with AflIII (shown by an arrow). (B) An expanded (GAA)230 repeat did not cause replication stalling of the T-antigen-driven replication. 2D gel electrophoresis of the replication intermediates of pUCneoGAA/CTT230 isolated from COS-1 cells and digested with AflIII and DpnI. (C) Replication stalling at the GAA repeat decreased over time. 2D gel electrophoresis of replication intermediates of the pUCneoGAA57 plasmid isolated at 2, 12, 18, 24 and 30 h after COS-1 cells transfection. Only gel-purified monomer plasmid was used for transfection. Replication intermediates were digested with AflIII. The descending part of the Y arc increased, while the bulges and the double Y arc decreased with incubation time.
Mentions: Our previous analysis of the alternative replication mode revealed that it is limited to the first replication cycle after episomal transfection into mammalian cells (26). We believe that the pattern of stalling at GAA repeat that we observed (Figures 2–5) was a superposition of the first replication cycle that initiated randomly throughout the sequence, and SV40 origin-initiated replication. Since the first cycle replication initiated everywhere, it mostly produced Y arc, and some of the bubble arc that we could only observe at overexposed pictures (Figure 5A, overexposed panel). During the first replication cycle, replication intermediates consist of the template DNA strands that came from Escherichia coli plasmid and nascent strands synthesized in mammalian cells. In subsequent replication cycles, both nascent and template strands are of mammalian origin. This allows one to distinguish between the first and the subsequent replication cycles using a DpnI restriction digest. This enzyme cleaves GATC sequences when they are methylated or hemimethylated by the bacterial Dam methylase. Thus, the DpnI digest eliminates the non-replicated episomal DNA, as well as the products of its first replication cycle in mammalian cells.Figure 5.

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