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Secondary structure formation and DNA instability at fragile site FRA16B.

Burrow AA, Marullo A, Holder LR, Wang YH - Nucleic Acids Res. (2010)

Bottom Line: Here, we show that FRA16B forms an alternative DNA structure in vitro.During replication in human cells, FRA16B exhibited reduced replication efficiency and expansions and deletions, depending on replication orientation and distance from the origin.These results strongly suggest that the secondary-structure-forming ability of FRA16B contributes to its fragility by stalling DNA replication, and this mechanism may be shared among other fragile DNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1016, USA.

ABSTRACT
Human chromosomal fragile sites are specific loci that are especially susceptible to DNA breakage following conditions of partial replication stress. They often are found in genes involved in tumorigenesis and map to over half of all known cancer-specific recurrent translocation breakpoints. While their molecular basis remains elusive, most fragile DNAs contain AT-rich flexibility islands predicted to form stable secondary structures. To understand the mechanism of fragile site instability, we examined the contribution of secondary structure formation to breakage at FRA16B. Here, we show that FRA16B forms an alternative DNA structure in vitro. During replication in human cells, FRA16B exhibited reduced replication efficiency and expansions and deletions, depending on replication orientation and distance from the origin. Furthermore, the examination of a FRA16B replication fork template demonstrated that the majority of the constructs contained DNA polymerase paused within the FRA16B sequence, and among the molecules, which completed DNA synthesis, 81% of them underwent fork reversal. These results strongly suggest that the secondary-structure-forming ability of FRA16B contributes to its fragility by stalling DNA replication, and this mechanism may be shared among other fragile DNAs.

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Secondary structure formation of FRA16B DNA following denaturation and re-annealing (reduplexing) reaction. (A) Gel electrophoresis analysis of re-annealed FRA16B DNA. DNA fragments of FRA16B (right) and pGEM3zf(+) (left) were subjected to reduplexing in increasing salt concentrations (0.1–1 M) and analyzed by native 4% PAGE. C, untreated samples; M, molecular weight marker. (B) Visualization of reduplexed FRA16B DNA by EM. After reduplexing reactions, samples were directly mounted onto carbon-coated copper EM grids and rotary shadowcasted with tungsten. Images are shown in reverse contrast. The total lengths of 100 molecules from each sample were measured using Image J software (lower right).
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Figure 1: Secondary structure formation of FRA16B DNA following denaturation and re-annealing (reduplexing) reaction. (A) Gel electrophoresis analysis of re-annealed FRA16B DNA. DNA fragments of FRA16B (right) and pGEM3zf(+) (left) were subjected to reduplexing in increasing salt concentrations (0.1–1 M) and analyzed by native 4% PAGE. C, untreated samples; M, molecular weight marker. (B) Visualization of reduplexed FRA16B DNA by EM. After reduplexing reactions, samples were directly mounted onto carbon-coated copper EM grids and rotary shadowcasted with tungsten. Images are shown in reverse contrast. The total lengths of 100 molecules from each sample were measured using Image J software (lower right).

Mentions: Since there has been no physical evidence of alternative DNA structures formed for AT-rich fragile DNAs, we investigated secondary structure formation by subjecting a 921-bp fragment of FRA16B to reduplexing in the presence of various concentrations of NaCl to allow re-annealing of the single strands following denaturation. Separation by polyacrylamide gel electrophoresis (PAGE) showed that the reduplexed FRA16B fragment gave rise to two slower-migrating products over a range of NaCl concentrations (Figure 1A). These products were not present in the untreated FRA16B sample or the reduplexed pGEM3zf(+) control, suggesting the formation of a secondary structure during reduplexing of FRA16B DNA. Furthermore, when FRA16B was denatured and only one strand of the duplex was labeled with 32P, the labeled strand corresponded uniquely to one of the bands with reduced electrophoretic mobility, demonstrating that each reduplexed band is produced from one of the two strands of FRA16B (Supplementary Figure S2). The examination of these molecules by EM confirmed that FRA16B folded into branched duplexes following denaturation and re-annealing (Figure 1B). These structures were not observed in the untreated FRA16B or reduplexed pGEM3zf(+) samples. The lengths of 100 DNA molecules from each sample were measured, and the comparison revealed that the reduplexed FRA16B molecules were shorter than the untreated FRA16B DNAs, indicating the presence of slipped-out regions participating in the formation of secondary structure. These data are the first to demonstrate that FRA16B is indeed able to form an alternative DNA structure in vitro.


Secondary structure formation and DNA instability at fragile site FRA16B.

Burrow AA, Marullo A, Holder LR, Wang YH - Nucleic Acids Res. (2010)

Secondary structure formation of FRA16B DNA following denaturation and re-annealing (reduplexing) reaction. (A) Gel electrophoresis analysis of re-annealed FRA16B DNA. DNA fragments of FRA16B (right) and pGEM3zf(+) (left) were subjected to reduplexing in increasing salt concentrations (0.1–1 M) and analyzed by native 4% PAGE. C, untreated samples; M, molecular weight marker. (B) Visualization of reduplexed FRA16B DNA by EM. After reduplexing reactions, samples were directly mounted onto carbon-coated copper EM grids and rotary shadowcasted with tungsten. Images are shown in reverse contrast. The total lengths of 100 molecules from each sample were measured using Image J software (lower right).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2875025&req=5

Figure 1: Secondary structure formation of FRA16B DNA following denaturation and re-annealing (reduplexing) reaction. (A) Gel electrophoresis analysis of re-annealed FRA16B DNA. DNA fragments of FRA16B (right) and pGEM3zf(+) (left) were subjected to reduplexing in increasing salt concentrations (0.1–1 M) and analyzed by native 4% PAGE. C, untreated samples; M, molecular weight marker. (B) Visualization of reduplexed FRA16B DNA by EM. After reduplexing reactions, samples were directly mounted onto carbon-coated copper EM grids and rotary shadowcasted with tungsten. Images are shown in reverse contrast. The total lengths of 100 molecules from each sample were measured using Image J software (lower right).
Mentions: Since there has been no physical evidence of alternative DNA structures formed for AT-rich fragile DNAs, we investigated secondary structure formation by subjecting a 921-bp fragment of FRA16B to reduplexing in the presence of various concentrations of NaCl to allow re-annealing of the single strands following denaturation. Separation by polyacrylamide gel electrophoresis (PAGE) showed that the reduplexed FRA16B fragment gave rise to two slower-migrating products over a range of NaCl concentrations (Figure 1A). These products were not present in the untreated FRA16B sample or the reduplexed pGEM3zf(+) control, suggesting the formation of a secondary structure during reduplexing of FRA16B DNA. Furthermore, when FRA16B was denatured and only one strand of the duplex was labeled with 32P, the labeled strand corresponded uniquely to one of the bands with reduced electrophoretic mobility, demonstrating that each reduplexed band is produced from one of the two strands of FRA16B (Supplementary Figure S2). The examination of these molecules by EM confirmed that FRA16B folded into branched duplexes following denaturation and re-annealing (Figure 1B). These structures were not observed in the untreated FRA16B or reduplexed pGEM3zf(+) samples. The lengths of 100 DNA molecules from each sample were measured, and the comparison revealed that the reduplexed FRA16B molecules were shorter than the untreated FRA16B DNAs, indicating the presence of slipped-out regions participating in the formation of secondary structure. These data are the first to demonstrate that FRA16B is indeed able to form an alternative DNA structure in vitro.

Bottom Line: Here, we show that FRA16B forms an alternative DNA structure in vitro.During replication in human cells, FRA16B exhibited reduced replication efficiency and expansions and deletions, depending on replication orientation and distance from the origin.These results strongly suggest that the secondary-structure-forming ability of FRA16B contributes to its fragility by stalling DNA replication, and this mechanism may be shared among other fragile DNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1016, USA.

ABSTRACT
Human chromosomal fragile sites are specific loci that are especially susceptible to DNA breakage following conditions of partial replication stress. They often are found in genes involved in tumorigenesis and map to over half of all known cancer-specific recurrent translocation breakpoints. While their molecular basis remains elusive, most fragile DNAs contain AT-rich flexibility islands predicted to form stable secondary structures. To understand the mechanism of fragile site instability, we examined the contribution of secondary structure formation to breakage at FRA16B. Here, we show that FRA16B forms an alternative DNA structure in vitro. During replication in human cells, FRA16B exhibited reduced replication efficiency and expansions and deletions, depending on replication orientation and distance from the origin. Furthermore, the examination of a FRA16B replication fork template demonstrated that the majority of the constructs contained DNA polymerase paused within the FRA16B sequence, and among the molecules, which completed DNA synthesis, 81% of them underwent fork reversal. These results strongly suggest that the secondary-structure-forming ability of FRA16B contributes to its fragility by stalling DNA replication, and this mechanism may be shared among other fragile DNAs.

Show MeSH
Related in: MedlinePlus