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The contribution of alu elements to mutagenic DNA double-strand break repair.

Morales ME, White TB, Streva VA, DeFreece CB, Hedges DJ, Deininger PL - PLoS Genet. (2015)

Bottom Line: Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence.This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences).Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events.

View Article: PubMed Central - PubMed

Affiliation: Tulane Cancer Center and Department of Epidemiology, Tulane University Health Sciences Center, New Orleans, Louisiana, United States of America.

ABSTRACT
Alu elements make up the largest family of human mobile elements, numbering 1.1 million copies and comprising 11% of the human genome. As a consequence of evolution and genetic drift, Alu elements of various sequence divergence exist throughout the human genome. Alu/Alu recombination has been shown to cause approximately 0.5% of new human genetic diseases and contribute to extensive genomic structural variation. To begin understanding the molecular mechanisms leading to these rearrangements in mammalian cells, we constructed Alu/Alu recombination reporter cell lines containing Alu elements ranging in sequence divergence from 0%-30% that allow detection of both Alu/Alu recombination and large non-homologous end joining (NHEJ) deletions that range from 1.0 to 1.9 kb in size. Introduction of as little as 0.7% sequence divergence between Alu elements resulted in a significant reduction in recombination, which indicates even small degrees of sequence divergence reduce the efficiency of homology-directed DNA double-strand break (DSB) repair. Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence. With greater levels of sequence divergence (15%-30%), we observed a significant increase in DSB repair due to a shift from Alu/Alu recombination to variable-length NHEJ which removes sequence between the two Alu elements. This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences). Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events. This is the first extensive study characterizing the influence of Alu element sequence divergence on DNA repair, which will inform predictions regarding the effect of Alu element sequence divergence on both the rate and nature of DNA repair events.

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Related in: MedlinePlus

The distribution of Alu/Alu recombination junctions in AARP HEK293FRT cells.(A) A schematic of the Alu/Alu recombination product indicates, in purple, a 5’ section of Alu1 (containing sequence divergence relative to Alu2) and, in pink, Alu 2 (Alu Ya5 consensus sequence). Arrows represent PCR primers used to amplify Alu/Alu recombination products (S2 Table). (B) The distribution of Alu/Alu recombination junctions in 3%-, 5%-, and 10%-AARP HEK293FRT cells as determined by PCR and sequence analysis of DNA repair products from isolated puror colonies. The Alu/Alu recombination product is divided into three segments of equal length (100 bp), which each contain the same extent of sequence divergence. The number of Alu/Alu recombination junctions expected and observed in each 100 bp interval is shown. An asterisk (*) marks an interval in which p<0.05 significance as determined by a chi-square test for observed vs. expected. (C-E) Alu/Alu recombination junctions mapped individually for the 3%- (C), 5%- (D), and 10%- (E) AARP HEK293FRT cells. The Alu/Alu recombination product was divided into segments of equal length according to the intervals of homology in which the Alu/Alu recombination junction can be mapped for each diverged AARP construct. The number of Alu/Alu recombination junctions observed is plotted on the y-axis for each interval of homology.
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pgen.1005016.g003: The distribution of Alu/Alu recombination junctions in AARP HEK293FRT cells.(A) A schematic of the Alu/Alu recombination product indicates, in purple, a 5’ section of Alu1 (containing sequence divergence relative to Alu2) and, in pink, Alu 2 (Alu Ya5 consensus sequence). Arrows represent PCR primers used to amplify Alu/Alu recombination products (S2 Table). (B) The distribution of Alu/Alu recombination junctions in 3%-, 5%-, and 10%-AARP HEK293FRT cells as determined by PCR and sequence analysis of DNA repair products from isolated puror colonies. The Alu/Alu recombination product is divided into three segments of equal length (100 bp), which each contain the same extent of sequence divergence. The number of Alu/Alu recombination junctions expected and observed in each 100 bp interval is shown. An asterisk (*) marks an interval in which p<0.05 significance as determined by a chi-square test for observed vs. expected. (C-E) Alu/Alu recombination junctions mapped individually for the 3%- (C), 5%- (D), and 10%- (E) AARP HEK293FRT cells. The Alu/Alu recombination product was divided into segments of equal length according to the intervals of homology in which the Alu/Alu recombination junction can be mapped for each diverged AARP construct. The number of Alu/Alu recombination junctions observed is plotted on the y-axis for each interval of homology.

Mentions: Several studies have shown that Alu/Alu recombination junctions naturally show a preference within the 5’ half of the Alu element [18–20]. To determine if Alu/Alu recombination junctions in our system similarly cluster to a particular region of the participating Alu elements, we mapped the recombination junctions of169 Alu/Alu recombination events from diverged AARP HEK293FRT cells (Fig. 3A). The Alu/Alu recombination product (chimeric Alu element) was divided into three 100 bp segments and the number of observed Alu/Alu recombination events in each interval was compared to the expected count if the recombination events occurred at random throughout the participating Alu elements (Fig. 3B). We observed a statistically significant increase in the number of recombination junctions occurring within the first 100 bp of the Alu element when compared to the remaining portion of the Alu. This is consistent with previous reports of genomic Alu elements involved in Alu/Alu recombination that suggest the presence of preferential recombination in the 5’ end of Alu [18,21]. We next wanted to assess the distribution of the Alu/Alu recombination junctions in the 3%-AARP, 5%-AARP, and 10%-AARP HEK293FRT cells separately. While the junctions in 3%-AARP cells appear evenly distributed (Fig. 3C), there is a clear bias toward the first 100 bp of the Alu element in 5%-AARP and 10%-AARP cells (Fig. 3D and 3E). To determine if the preference for the formation of a recombination junction within the Alu element was mediated by the length of the region of perfect homology, we modified the 5%-AARP constructs to include two longer stretches of homology, 40 bp, either at each end (S9A Fig) or in the middle of the Alu element (S9B Fig) and a construct with 100 bp of homology in the middle of the Alu element (S9C Fig). Surprisingly, introduction of increased lengths of perfect sequence homology did not shift the Alu/Alu recombination junction preference from the first 100 bp of the Alu element (S9 Fig). Thus, although the overall sequence homology between Alu elements clearly contributes to the rate of Alu/Alu recombination, the actual resolution of the repair junction does not appear to be directed to regions of longer homology.


The contribution of alu elements to mutagenic DNA double-strand break repair.

Morales ME, White TB, Streva VA, DeFreece CB, Hedges DJ, Deininger PL - PLoS Genet. (2015)

The distribution of Alu/Alu recombination junctions in AARP HEK293FRT cells.(A) A schematic of the Alu/Alu recombination product indicates, in purple, a 5’ section of Alu1 (containing sequence divergence relative to Alu2) and, in pink, Alu 2 (Alu Ya5 consensus sequence). Arrows represent PCR primers used to amplify Alu/Alu recombination products (S2 Table). (B) The distribution of Alu/Alu recombination junctions in 3%-, 5%-, and 10%-AARP HEK293FRT cells as determined by PCR and sequence analysis of DNA repair products from isolated puror colonies. The Alu/Alu recombination product is divided into three segments of equal length (100 bp), which each contain the same extent of sequence divergence. The number of Alu/Alu recombination junctions expected and observed in each 100 bp interval is shown. An asterisk (*) marks an interval in which p<0.05 significance as determined by a chi-square test for observed vs. expected. (C-E) Alu/Alu recombination junctions mapped individually for the 3%- (C), 5%- (D), and 10%- (E) AARP HEK293FRT cells. The Alu/Alu recombination product was divided into segments of equal length according to the intervals of homology in which the Alu/Alu recombination junction can be mapped for each diverged AARP construct. The number of Alu/Alu recombination junctions observed is plotted on the y-axis for each interval of homology.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4356517&req=5

pgen.1005016.g003: The distribution of Alu/Alu recombination junctions in AARP HEK293FRT cells.(A) A schematic of the Alu/Alu recombination product indicates, in purple, a 5’ section of Alu1 (containing sequence divergence relative to Alu2) and, in pink, Alu 2 (Alu Ya5 consensus sequence). Arrows represent PCR primers used to amplify Alu/Alu recombination products (S2 Table). (B) The distribution of Alu/Alu recombination junctions in 3%-, 5%-, and 10%-AARP HEK293FRT cells as determined by PCR and sequence analysis of DNA repair products from isolated puror colonies. The Alu/Alu recombination product is divided into three segments of equal length (100 bp), which each contain the same extent of sequence divergence. The number of Alu/Alu recombination junctions expected and observed in each 100 bp interval is shown. An asterisk (*) marks an interval in which p<0.05 significance as determined by a chi-square test for observed vs. expected. (C-E) Alu/Alu recombination junctions mapped individually for the 3%- (C), 5%- (D), and 10%- (E) AARP HEK293FRT cells. The Alu/Alu recombination product was divided into segments of equal length according to the intervals of homology in which the Alu/Alu recombination junction can be mapped for each diverged AARP construct. The number of Alu/Alu recombination junctions observed is plotted on the y-axis for each interval of homology.
Mentions: Several studies have shown that Alu/Alu recombination junctions naturally show a preference within the 5’ half of the Alu element [18–20]. To determine if Alu/Alu recombination junctions in our system similarly cluster to a particular region of the participating Alu elements, we mapped the recombination junctions of169 Alu/Alu recombination events from diverged AARP HEK293FRT cells (Fig. 3A). The Alu/Alu recombination product (chimeric Alu element) was divided into three 100 bp segments and the number of observed Alu/Alu recombination events in each interval was compared to the expected count if the recombination events occurred at random throughout the participating Alu elements (Fig. 3B). We observed a statistically significant increase in the number of recombination junctions occurring within the first 100 bp of the Alu element when compared to the remaining portion of the Alu. This is consistent with previous reports of genomic Alu elements involved in Alu/Alu recombination that suggest the presence of preferential recombination in the 5’ end of Alu [18,21]. We next wanted to assess the distribution of the Alu/Alu recombination junctions in the 3%-AARP, 5%-AARP, and 10%-AARP HEK293FRT cells separately. While the junctions in 3%-AARP cells appear evenly distributed (Fig. 3C), there is a clear bias toward the first 100 bp of the Alu element in 5%-AARP and 10%-AARP cells (Fig. 3D and 3E). To determine if the preference for the formation of a recombination junction within the Alu element was mediated by the length of the region of perfect homology, we modified the 5%-AARP constructs to include two longer stretches of homology, 40 bp, either at each end (S9A Fig) or in the middle of the Alu element (S9B Fig) and a construct with 100 bp of homology in the middle of the Alu element (S9C Fig). Surprisingly, introduction of increased lengths of perfect sequence homology did not shift the Alu/Alu recombination junction preference from the first 100 bp of the Alu element (S9 Fig). Thus, although the overall sequence homology between Alu elements clearly contributes to the rate of Alu/Alu recombination, the actual resolution of the repair junction does not appear to be directed to regions of longer homology.

Bottom Line: Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence.This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences).Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events.

View Article: PubMed Central - PubMed

Affiliation: Tulane Cancer Center and Department of Epidemiology, Tulane University Health Sciences Center, New Orleans, Louisiana, United States of America.

ABSTRACT
Alu elements make up the largest family of human mobile elements, numbering 1.1 million copies and comprising 11% of the human genome. As a consequence of evolution and genetic drift, Alu elements of various sequence divergence exist throughout the human genome. Alu/Alu recombination has been shown to cause approximately 0.5% of new human genetic diseases and contribute to extensive genomic structural variation. To begin understanding the molecular mechanisms leading to these rearrangements in mammalian cells, we constructed Alu/Alu recombination reporter cell lines containing Alu elements ranging in sequence divergence from 0%-30% that allow detection of both Alu/Alu recombination and large non-homologous end joining (NHEJ) deletions that range from 1.0 to 1.9 kb in size. Introduction of as little as 0.7% sequence divergence between Alu elements resulted in a significant reduction in recombination, which indicates even small degrees of sequence divergence reduce the efficiency of homology-directed DNA double-strand break (DSB) repair. Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence. With greater levels of sequence divergence (15%-30%), we observed a significant increase in DSB repair due to a shift from Alu/Alu recombination to variable-length NHEJ which removes sequence between the two Alu elements. This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences). Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events. This is the first extensive study characterizing the influence of Alu element sequence divergence on DNA repair, which will inform predictions regarding the effect of Alu element sequence divergence on both the rate and nature of DNA repair events.

Show MeSH
Related in: MedlinePlus