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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 pUCneoGAA/CTTn plasmid. (GAA)n repeat was inserted in pUCneo in two orientations. Replication intermediates were digested by AflIII. The hybridization probe used in 2D gel analysis corresponded to the GAA repeat-containing AflIII fragment. (B) Schematic presentation of the 2D gel electrophoresis of mammalian replication intermediates digested with AflIII. Corresponding shapes of replication intermediates are shown in gray. Spot 0 – unreplicated AflIII fragment; spot 1 – replication stalling of the counterclockwise replication fork at GAA; spot 2 – replication stalling of the clockwise replication fork at GAA; spot 3 – two replication forks from opposite ends of the fragment stalled at the repeat. May also contain intermolecular complexes of two different fragments joined at the GAA repeat. A short stretch of the bubble arc was omitted from this scheme because it is obscured by unreplicated DNA spot in our 2D gel pictures.
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gks021-F1: (A) The scheme of pUCneoGAA/CTTn plasmid. (GAA)n repeat was inserted in pUCneo in two orientations. Replication intermediates were digested by AflIII. The hybridization probe used in 2D gel analysis corresponded to the GAA repeat-containing AflIII fragment. (B) Schematic presentation of the 2D gel electrophoresis of mammalian replication intermediates digested with AflIII. Corresponding shapes of replication intermediates are shown in gray. Spot 0 – unreplicated AflIII fragment; spot 1 – replication stalling of the counterclockwise replication fork at GAA; spot 2 – replication stalling of the clockwise replication fork at GAA; spot 3 – two replication forks from opposite ends of the fragment stalled at the repeat. May also contain intermolecular complexes of two different fragments joined at the GAA repeat. A short stretch of the bubble arc was omitted from this scheme because it is obscured by unreplicated DNA spot in our 2D gel pictures.

Mentions: We analyzed the progression of DNA replication through GAA repeats of varying lengths inserted into a pSV2neo-derived plasmid, pUCneo (Figure 1A). The resultant plasmids were introduced into T-antigen-expressing COS-1 monkey fibroblasts by transient transfection. The repeats were positioned on the path of the counterclockwise replication fork initiated at the episomal SV40 origin. We carefully controlled the quality of DNA samples used for transfection to assure that they mainly contained monomeric plasmids. Thirty hours after transfection into mammalian cells, the plasmids and their replication intermediates were isolated. The replication intermediates were digested with the AflIII restriction endonuclease, which generated a fragment with a GAA repeat positioned at approximately one-third of a distance from its end. The replication intermediated were then analyzed by 2D agarose gel electrophoresis followed by Southern hybridization (Figure 2).Figure 1.


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 pUCneoGAA/CTTn plasmid. (GAA)n repeat was inserted in pUCneo in two orientations. Replication intermediates were digested by AflIII. The hybridization probe used in 2D gel analysis corresponded to the GAA repeat-containing AflIII fragment. (B) Schematic presentation of the 2D gel electrophoresis of mammalian replication intermediates digested with AflIII. Corresponding shapes of replication intermediates are shown in gray. Spot 0 – unreplicated AflIII fragment; spot 1 – replication stalling of the counterclockwise replication fork at GAA; spot 2 – replication stalling of the clockwise replication fork at GAA; spot 3 – two replication forks from opposite ends of the fragment stalled at the repeat. May also contain intermolecular complexes of two different fragments joined at the GAA repeat. A short stretch of the bubble arc was omitted from this scheme because it is obscured by unreplicated DNA spot in our 2D gel pictures.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks021-F1: (A) The scheme of pUCneoGAA/CTTn plasmid. (GAA)n repeat was inserted in pUCneo in two orientations. Replication intermediates were digested by AflIII. The hybridization probe used in 2D gel analysis corresponded to the GAA repeat-containing AflIII fragment. (B) Schematic presentation of the 2D gel electrophoresis of mammalian replication intermediates digested with AflIII. Corresponding shapes of replication intermediates are shown in gray. Spot 0 – unreplicated AflIII fragment; spot 1 – replication stalling of the counterclockwise replication fork at GAA; spot 2 – replication stalling of the clockwise replication fork at GAA; spot 3 – two replication forks from opposite ends of the fragment stalled at the repeat. May also contain intermolecular complexes of two different fragments joined at the GAA repeat. A short stretch of the bubble arc was omitted from this scheme because it is obscured by unreplicated DNA spot in our 2D gel pictures.
Mentions: We analyzed the progression of DNA replication through GAA repeats of varying lengths inserted into a pSV2neo-derived plasmid, pUCneo (Figure 1A). The resultant plasmids were introduced into T-antigen-expressing COS-1 monkey fibroblasts by transient transfection. The repeats were positioned on the path of the counterclockwise replication fork initiated at the episomal SV40 origin. We carefully controlled the quality of DNA samples used for transfection to assure that they mainly contained monomeric plasmids. Thirty hours after transfection into mammalian cells, the plasmids and their replication intermediates were isolated. The replication intermediates were digested with the AflIII restriction endonuclease, which generated a fragment with a GAA repeat positioned at approximately one-third of a distance from its end. The replication intermediated were then analyzed by 2D agarose gel electrophoresis followed by Southern hybridization (Figure 2).Figure 1.

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