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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 scheme of the Ori(-)GAA/CTT57 plasmid. In the Ori(-) plasmid, the SV40 replication origin was replaced by the nuclear localization sequence (26). (B) 2D gel electrophoresis analysis of replication intermediates of Ori(-)GAA/CTT57, and Ori(-), isolated from Cos-1 cells. Replication of Ori(-) plasmid was blocked by the (GAA)57 repeat in both orientations. (C) 2D gel electrophoresis analysis of replication intermediates of nicked Ori(-)GAA57 plasmid isolated from Cos-1 cells. Replication of the nicked plasmid that did not contain preformed structures was still affected by GAA repeat. The positions of nicks introduced by Nt. BstNBI are shown by small circles in Figure 6A. We concluded that the GAA repeat forms an alternative structure that stalls replication inside the mammalian cell. (D) S1 nuclease digest of nicked and supercoiled Ori(-)GAA57 plasmids to confirm that alternative DNA structures do not form in the nicked Ori(-)GAA57. 1 – series of bands corresponding to the GAA repeat position that are visible only for the supercoiled sample; 2 – 957 bp ScaI–NdeI fragment; 3 – supercoiled pUCneo, 4 – open circle pUCneo. (E) 2D gel of the replication intermediates of pUCneoGAA57 isolated from 293A cells. Alternative replication of pUCneoGAA57 happens in the absence of T-antigen and is severely stalled by the GAA repeat. The stalling points are indicated by arrows.
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gks021-F6: (A) The scheme of the Ori(-)GAA/CTT57 plasmid. In the Ori(-) plasmid, the SV40 replication origin was replaced by the nuclear localization sequence (26). (B) 2D gel electrophoresis analysis of replication intermediates of Ori(-)GAA/CTT57, and Ori(-), isolated from Cos-1 cells. Replication of Ori(-) plasmid was blocked by the (GAA)57 repeat in both orientations. (C) 2D gel electrophoresis analysis of replication intermediates of nicked Ori(-)GAA57 plasmid isolated from Cos-1 cells. Replication of the nicked plasmid that did not contain preformed structures was still affected by GAA repeat. The positions of nicks introduced by Nt. BstNBI are shown by small circles in Figure 6A. We concluded that the GAA repeat forms an alternative structure that stalls replication inside the mammalian cell. (D) S1 nuclease digest of nicked and supercoiled Ori(-)GAA57 plasmids to confirm that alternative DNA structures do not form in the nicked Ori(-)GAA57. 1 – series of bands corresponding to the GAA repeat position that are visible only for the supercoiled sample; 2 – 957 bp ScaI–NdeI fragment; 3 – supercoiled pUCneo, 4 – open circle pUCneo. (E) 2D gel of the replication intermediates of pUCneoGAA57 isolated from 293A cells. Alternative replication of pUCneoGAA57 happens in the absence of T-antigen and is severely stalled by the GAA repeat. The stalling points are indicated by arrows.

Mentions: To explore whether replication stalling occurred in the course of the alternative replication, we analyzed the replication of the Ori(-)GAA57 plasmid, which did not contain the SV40 replication origin (Figure 6A). One can see that in COS-1 cells this plasmid was still capable of undergoing some replication, which was characterized by the profound fork stalling at the GAA repeat (Figure 6B). The fact that the descending portion of Y arc beyond the spot 2 was not detectable in the Ori(-) plasmids is consistent with the complete replication blockage at GAA repeat.Figure 6.


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 scheme of the Ori(-)GAA/CTT57 plasmid. In the Ori(-) plasmid, the SV40 replication origin was replaced by the nuclear localization sequence (26). (B) 2D gel electrophoresis analysis of replication intermediates of Ori(-)GAA/CTT57, and Ori(-), isolated from Cos-1 cells. Replication of Ori(-) plasmid was blocked by the (GAA)57 repeat in both orientations. (C) 2D gel electrophoresis analysis of replication intermediates of nicked Ori(-)GAA57 plasmid isolated from Cos-1 cells. Replication of the nicked plasmid that did not contain preformed structures was still affected by GAA repeat. The positions of nicks introduced by Nt. BstNBI are shown by small circles in Figure 6A. We concluded that the GAA repeat forms an alternative structure that stalls replication inside the mammalian cell. (D) S1 nuclease digest of nicked and supercoiled Ori(-)GAA57 plasmids to confirm that alternative DNA structures do not form in the nicked Ori(-)GAA57. 1 – series of bands corresponding to the GAA repeat position that are visible only for the supercoiled sample; 2 – 957 bp ScaI–NdeI fragment; 3 – supercoiled pUCneo, 4 – open circle pUCneo. (E) 2D gel of the replication intermediates of pUCneoGAA57 isolated from 293A cells. Alternative replication of pUCneoGAA57 happens in the absence of T-antigen and is severely stalled by the GAA repeat. The stalling points are indicated by arrows.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks021-F6: (A) The scheme of the Ori(-)GAA/CTT57 plasmid. In the Ori(-) plasmid, the SV40 replication origin was replaced by the nuclear localization sequence (26). (B) 2D gel electrophoresis analysis of replication intermediates of Ori(-)GAA/CTT57, and Ori(-), isolated from Cos-1 cells. Replication of Ori(-) plasmid was blocked by the (GAA)57 repeat in both orientations. (C) 2D gel electrophoresis analysis of replication intermediates of nicked Ori(-)GAA57 plasmid isolated from Cos-1 cells. Replication of the nicked plasmid that did not contain preformed structures was still affected by GAA repeat. The positions of nicks introduced by Nt. BstNBI are shown by small circles in Figure 6A. We concluded that the GAA repeat forms an alternative structure that stalls replication inside the mammalian cell. (D) S1 nuclease digest of nicked and supercoiled Ori(-)GAA57 plasmids to confirm that alternative DNA structures do not form in the nicked Ori(-)GAA57. 1 – series of bands corresponding to the GAA repeat position that are visible only for the supercoiled sample; 2 – 957 bp ScaI–NdeI fragment; 3 – supercoiled pUCneo, 4 – open circle pUCneo. (E) 2D gel of the replication intermediates of pUCneoGAA57 isolated from 293A cells. Alternative replication of pUCneoGAA57 happens in the absence of T-antigen and is severely stalled by the GAA repeat. The stalling points are indicated by arrows.
Mentions: To explore whether replication stalling occurred in the course of the alternative replication, we analyzed the replication of the Ori(-)GAA57 plasmid, which did not contain the SV40 replication origin (Figure 6A). One can see that in COS-1 cells this plasmid was still capable of undergoing some replication, which was characterized by the profound fork stalling at the GAA repeat (Figure 6B). The fact that the descending portion of Y arc beyond the spot 2 was not detectable in the Ori(-) plasmids is consistent with the complete replication blockage at GAA repeat.Figure 6.

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