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Heterozygosity for a hypomorphic Polβ mutation reduces the expansion frequency in a mouse model of the Fragile X-related disorders.

Lokanga RA, Senejani AG, Sweasy JB, Usdin K - PLoS Genet. (2015)

Bottom Line: The FXDs result from expansion of a CGG/CCG repeat tract in the 5' UTR of the FMR1 gene.Somewhat surprisingly, while the number of expansions is smaller, the average size of the residual expansions is larger than that seen in WT animals.This may have interesting implications for the mechanism by which BER generates expansions.

View Article: PubMed Central - PubMed

Affiliation: Section on Gene Structure and Disease, Laboratory of Cell and molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America; Department of Biochemistry, University of Cape Town Medical School, Cape Town, South Africa.

ABSTRACT
The Fragile X-related disorders (FXDs) are members of the Repeat Expansion Diseases, a group of human genetic conditions resulting from expansion of a specific tandem repeat. The FXDs result from expansion of a CGG/CCG repeat tract in the 5' UTR of the FMR1 gene. While expansion in a FXD mouse model is known to require some mismatch repair (MMR) proteins, our previous work and work in mouse models of another Repeat Expansion Disease show that early events in the base excision repair (BER) pathway play a role in the expansion process. One model for repeat expansion proposes that a non-canonical MMR process makes use of the nicks generated early in BER to load the MMR machinery that then generates expansions. However, we show here that heterozygosity for a Y265C mutation in Polβ, a key polymerase in the BER pathway, is enough to significantly reduce both the number of expansions seen in paternal gametes and the extent of somatic expansion in some tissues of the FXD mouse. These data suggest that events in the BER pathway downstream of the generation of nicks are also important for repeat expansion. Somewhat surprisingly, while the number of expansions is smaller, the average size of the residual expansions is larger than that seen in WT animals. This may have interesting implications for the mechanism by which BER generates expansions.

No MeSH data available.


Related in: MedlinePlus

Model for BER-mediated repeat expansion in the FX PM mouse.Nicks that do not get repaired by short patch BER may be channeled into one of two branches of the LP BER pathway. (i) The Polβ-dependent, Polδ/Polε-independent branch. Nick processing is carried out by Polβ, a poorly processive polymerase with weak strand-displacement activity. The resultant small flaps are processed by FEN1 to generate a ligatable 5’ end that still contains a few additional flap bases (shown in orange). (ii) The Polβ/Polδ/Polε-dependent branch. Since both Polδ and Polε are more processive than Polβ, more strand slippage and more extensive strand displacement may result. Repriming of DNA synthesis on the slipped-strand using Polβ would not remove looped out bases (shown in red) since Polβ lacks a suitable proofreading activity. Limited strand displacement by Polβ or more extensive strand displacement by Polδ/Polε, followed by FEN1 cleavage could also result a ligatable end that still contains some flap bases (shown in orange). In either case, repair synthesis initiated on the complementary strand would fix the supernumerary bases into the derivative allele thus generating either a small (i) or large (ii) expansion. MMR proteins may facilitate this process by stabilizing the hairpins. These proteins may also directly generate expansions by channelling the hairpins formed during BER into the MMR pathway.
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pgen.1005181.g007: Model for BER-mediated repeat expansion in the FX PM mouse.Nicks that do not get repaired by short patch BER may be channeled into one of two branches of the LP BER pathway. (i) The Polβ-dependent, Polδ/Polε-independent branch. Nick processing is carried out by Polβ, a poorly processive polymerase with weak strand-displacement activity. The resultant small flaps are processed by FEN1 to generate a ligatable 5’ end that still contains a few additional flap bases (shown in orange). (ii) The Polβ/Polδ/Polε-dependent branch. Since both Polδ and Polε are more processive than Polβ, more strand slippage and more extensive strand displacement may result. Repriming of DNA synthesis on the slipped-strand using Polβ would not remove looped out bases (shown in red) since Polβ lacks a suitable proofreading activity. Limited strand displacement by Polβ or more extensive strand displacement by Polδ/Polε, followed by FEN1 cleavage could also result a ligatable end that still contains some flap bases (shown in orange). In either case, repair synthesis initiated on the complementary strand would fix the supernumerary bases into the derivative allele thus generating either a small (i) or large (ii) expansion. MMR proteins may facilitate this process by stabilizing the hairpins. These proteins may also directly generate expansions by channelling the hairpins formed during BER into the MMR pathway.

Mentions: One proposed model for BER-mediated repeat expansion suggests that expansion results from a Polβ/FEN1-dependent branch of the LP BER pathway where the weak strand displacement synthesis activity of Polβ is proposed to facilitate limited strand-slippage of the repeats in the DNA downstream of the nick [42] as illustrated in Fig 7(i). This process would be promoted by hairpin formation by the repeats and would create a larger gap that would need to be filled by Polβ. A stable hairpin would force FEN1 to capture and cleave a series of short flaps resulting from breathing or realignment of the 5’ end of the hairpins. This so-called alternate cleavage process would be essential for the creation of the ligatable nick necessary to complete repair. The failure to fully remove the flap would result in expansions. A second model, illustrated in Fig 7(ii), proposes that expansion arises via the use of a second branch of the LP BER pathway in which DNA synthesis is carried out by a combination of Polβ, Polδ and perhaps Polε. Expansion would be triggered by strand slippage of the nascent strand during progressive DNA synthesis by Polδ/Polε and the resultant formation of a hairpin by the repeats [43]. If the hairpin does not have a 3’ tail, Polδ and Polε could reinitiate DNA synthesis after using their 3’-5’ proofreading activity to remove the hairpin. However, if Polβ reinitiates synthesis the hairpin would be retained since Polβ has no such proofreading activity. This would result in repeat expansion if the hairpin were not subsequently removed. Some strand displacement could also contribute to the incorporation of additional bases.


Heterozygosity for a hypomorphic Polβ mutation reduces the expansion frequency in a mouse model of the Fragile X-related disorders.

Lokanga RA, Senejani AG, Sweasy JB, Usdin K - PLoS Genet. (2015)

Model for BER-mediated repeat expansion in the FX PM mouse.Nicks that do not get repaired by short patch BER may be channeled into one of two branches of the LP BER pathway. (i) The Polβ-dependent, Polδ/Polε-independent branch. Nick processing is carried out by Polβ, a poorly processive polymerase with weak strand-displacement activity. The resultant small flaps are processed by FEN1 to generate a ligatable 5’ end that still contains a few additional flap bases (shown in orange). (ii) The Polβ/Polδ/Polε-dependent branch. Since both Polδ and Polε are more processive than Polβ, more strand slippage and more extensive strand displacement may result. Repriming of DNA synthesis on the slipped-strand using Polβ would not remove looped out bases (shown in red) since Polβ lacks a suitable proofreading activity. Limited strand displacement by Polβ or more extensive strand displacement by Polδ/Polε, followed by FEN1 cleavage could also result a ligatable end that still contains some flap bases (shown in orange). In either case, repair synthesis initiated on the complementary strand would fix the supernumerary bases into the derivative allele thus generating either a small (i) or large (ii) expansion. MMR proteins may facilitate this process by stabilizing the hairpins. These proteins may also directly generate expansions by channelling the hairpins formed during BER into the MMR pathway.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401650&req=5

pgen.1005181.g007: Model for BER-mediated repeat expansion in the FX PM mouse.Nicks that do not get repaired by short patch BER may be channeled into one of two branches of the LP BER pathway. (i) The Polβ-dependent, Polδ/Polε-independent branch. Nick processing is carried out by Polβ, a poorly processive polymerase with weak strand-displacement activity. The resultant small flaps are processed by FEN1 to generate a ligatable 5’ end that still contains a few additional flap bases (shown in orange). (ii) The Polβ/Polδ/Polε-dependent branch. Since both Polδ and Polε are more processive than Polβ, more strand slippage and more extensive strand displacement may result. Repriming of DNA synthesis on the slipped-strand using Polβ would not remove looped out bases (shown in red) since Polβ lacks a suitable proofreading activity. Limited strand displacement by Polβ or more extensive strand displacement by Polδ/Polε, followed by FEN1 cleavage could also result a ligatable end that still contains some flap bases (shown in orange). In either case, repair synthesis initiated on the complementary strand would fix the supernumerary bases into the derivative allele thus generating either a small (i) or large (ii) expansion. MMR proteins may facilitate this process by stabilizing the hairpins. These proteins may also directly generate expansions by channelling the hairpins formed during BER into the MMR pathway.
Mentions: One proposed model for BER-mediated repeat expansion suggests that expansion results from a Polβ/FEN1-dependent branch of the LP BER pathway where the weak strand displacement synthesis activity of Polβ is proposed to facilitate limited strand-slippage of the repeats in the DNA downstream of the nick [42] as illustrated in Fig 7(i). This process would be promoted by hairpin formation by the repeats and would create a larger gap that would need to be filled by Polβ. A stable hairpin would force FEN1 to capture and cleave a series of short flaps resulting from breathing or realignment of the 5’ end of the hairpins. This so-called alternate cleavage process would be essential for the creation of the ligatable nick necessary to complete repair. The failure to fully remove the flap would result in expansions. A second model, illustrated in Fig 7(ii), proposes that expansion arises via the use of a second branch of the LP BER pathway in which DNA synthesis is carried out by a combination of Polβ, Polδ and perhaps Polε. Expansion would be triggered by strand slippage of the nascent strand during progressive DNA synthesis by Polδ/Polε and the resultant formation of a hairpin by the repeats [43]. If the hairpin does not have a 3’ tail, Polδ and Polε could reinitiate DNA synthesis after using their 3’-5’ proofreading activity to remove the hairpin. However, if Polβ reinitiates synthesis the hairpin would be retained since Polβ has no such proofreading activity. This would result in repeat expansion if the hairpin were not subsequently removed. Some strand displacement could also contribute to the incorporation of additional bases.

Bottom Line: The FXDs result from expansion of a CGG/CCG repeat tract in the 5' UTR of the FMR1 gene.Somewhat surprisingly, while the number of expansions is smaller, the average size of the residual expansions is larger than that seen in WT animals.This may have interesting implications for the mechanism by which BER generates expansions.

View Article: PubMed Central - PubMed

Affiliation: Section on Gene Structure and Disease, Laboratory of Cell and molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America; Department of Biochemistry, University of Cape Town Medical School, Cape Town, South Africa.

ABSTRACT
The Fragile X-related disorders (FXDs) are members of the Repeat Expansion Diseases, a group of human genetic conditions resulting from expansion of a specific tandem repeat. The FXDs result from expansion of a CGG/CCG repeat tract in the 5' UTR of the FMR1 gene. While expansion in a FXD mouse model is known to require some mismatch repair (MMR) proteins, our previous work and work in mouse models of another Repeat Expansion Disease show that early events in the base excision repair (BER) pathway play a role in the expansion process. One model for repeat expansion proposes that a non-canonical MMR process makes use of the nicks generated early in BER to load the MMR machinery that then generates expansions. However, we show here that heterozygosity for a Y265C mutation in Polβ, a key polymerase in the BER pathway, is enough to significantly reduce both the number of expansions seen in paternal gametes and the extent of somatic expansion in some tissues of the FXD mouse. These data suggest that events in the BER pathway downstream of the generation of nicks are also important for repeat expansion. Somewhat surprisingly, while the number of expansions is smaller, the average size of the residual expansions is larger than that seen in WT animals. This may have interesting implications for the mechanism by which BER generates expansions.

No MeSH data available.


Related in: MedlinePlus