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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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Related in: MedlinePlus

Proposed models of FRA16B instability. (A) Replication fork demonstrating DNA polymerase (gray circle) entering the FRA16B sequence (bold). (B) During FRA16B replication, the polymerase has a high tendency to pause, likely at regions of secondary structure formation. Consequently, DNA strand breakage, deletions or insertions may arise. (C) FRA16B may promote spontaneous fork reversal, leading to a four-stranded chickenfoot intermediate. (D) FRA16B may form an extensive secondary structure on a template strand or (E) a newly synthesized strand, leading to deletions and insertions, respectively.
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Figure 4: Proposed models of FRA16B instability. (A) Replication fork demonstrating DNA polymerase (gray circle) entering the FRA16B sequence (bold). (B) During FRA16B replication, the polymerase has a high tendency to pause, likely at regions of secondary structure formation. Consequently, DNA strand breakage, deletions or insertions may arise. (C) FRA16B may promote spontaneous fork reversal, leading to a four-stranded chickenfoot intermediate. (D) FRA16B may form an extensive secondary structure on a template strand or (E) a newly synthesized strand, leading to deletions and insertions, respectively.

Mentions: This study is the first to provide evidence of spontaneous replication fork reversal within a fragile site sequence during DNA synthesis. Based on this information, we propose a model whereby FRA16B instability primarily arises from polymerase stalling caused by the formation of secondary structure at the fragile site region (Figure 4B), coupled with the failure of the ATR checkpoint pathway. Other mechanisms contributing to fragile site instability could be due to replication fork regression (Figure 4C), the formation of stable DNA secondary structures on the lagging strand template leading to polymerase skipping (Figure 4D) or replication restart with secondary structure formed on the newly synthesized strand (Figure 4E). These mechanisms may be shared among other fragile DNA sequences. Overall, our results provide insight into the mechanism of fragile site instability by demonstrating the ability of a fragile DNA to form a stable secondary structure that affects replication efficiency and instability, and causes polymerase pausing during DNA synthesis.Figure 4.


Secondary structure formation and DNA instability at fragile site FRA16B.

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

Proposed models of FRA16B instability. (A) Replication fork demonstrating DNA polymerase (gray circle) entering the FRA16B sequence (bold). (B) During FRA16B replication, the polymerase has a high tendency to pause, likely at regions of secondary structure formation. Consequently, DNA strand breakage, deletions or insertions may arise. (C) FRA16B may promote spontaneous fork reversal, leading to a four-stranded chickenfoot intermediate. (D) FRA16B may form an extensive secondary structure on a template strand or (E) a newly synthesized strand, leading to deletions and insertions, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875025&req=5

Figure 4: Proposed models of FRA16B instability. (A) Replication fork demonstrating DNA polymerase (gray circle) entering the FRA16B sequence (bold). (B) During FRA16B replication, the polymerase has a high tendency to pause, likely at regions of secondary structure formation. Consequently, DNA strand breakage, deletions or insertions may arise. (C) FRA16B may promote spontaneous fork reversal, leading to a four-stranded chickenfoot intermediate. (D) FRA16B may form an extensive secondary structure on a template strand or (E) a newly synthesized strand, leading to deletions and insertions, respectively.
Mentions: This study is the first to provide evidence of spontaneous replication fork reversal within a fragile site sequence during DNA synthesis. Based on this information, we propose a model whereby FRA16B instability primarily arises from polymerase stalling caused by the formation of secondary structure at the fragile site region (Figure 4B), coupled with the failure of the ATR checkpoint pathway. Other mechanisms contributing to fragile site instability could be due to replication fork regression (Figure 4C), the formation of stable DNA secondary structures on the lagging strand template leading to polymerase skipping (Figure 4D) or replication restart with secondary structure formed on the newly synthesized strand (Figure 4E). These mechanisms may be shared among other fragile DNA sequences. Overall, our results provide insight into the mechanism of fragile site instability by demonstrating the ability of a fragile DNA to form a stable secondary structure that affects replication efficiency and instability, and causes polymerase pausing during DNA synthesis.Figure 4.

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