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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 Alu/Alu recombination system.(A) Schematic of the Alu/Alu recombination reporter. Cells harboring the reporter were neomycin resistant (neor) and sensitive to puromycin (puros) or blasticidin (blasts) or eGFP negative (eGFP-), depending on the reporter cassette used (AARP, AARB or AARG). Cells were grown in the presence of neomycin to eliminate cells that underwent spurious recombination before induction of DNA double-strand breaks (DSBs). DSBs were induced by transfection of an I-SceI endonuclease expression vector. (B) The reporter cassette was inserted using the Flp-In system (see S1 Fig, Materials and Methods) into a single genomic location in each cell line, which allows variants of the vector (i.e. different Alu elements) to be inserted in the same chromosomal location. (C) Repair of an I-SceI-induced DNA DSB may result in a deletion of the sequence between the two Alu elements after which the cell becomes neomycin sensitive (neos) and express the puromycin resistance (puroR), blasticidin resistance (blastR) or eGFP (eGFP+) gene. (D and E) I-SceI-induced DNA DSBs in the AARP (D) and AARB (E) HEK293FRT cells result in Alu/Alu recombination. Cells were transiently transfected with either I-SceI expression vector or empty vector (pUC19) control. Puromycin resistant (puror) or blasticidin resistant (blastr) colonies were counted and are shown graphically. Error bars denote standard error and statistical significance was shown using one-way ANOVA. Below the graphs are representative flasks showing puror or blastr colonies following I-SceI-induced DNA DSBs. (F) I-SceI-induced DNA DSBs in 0%-AARG HEK293FRT cells result in eGFP+ cells. The percentage of eGFP+ cells are shown graphically. Error bars represent standard error and statistical significance was shown using Student’s t-test. Representative flow plots from fluorescence activated cell sorting (FACS) analysis following transfections with I-SceI or empty vector pUC19 are shown below the graphs.
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pgen.1005016.g001: The Alu/Alu recombination system.(A) Schematic of the Alu/Alu recombination reporter. Cells harboring the reporter were neomycin resistant (neor) and sensitive to puromycin (puros) or blasticidin (blasts) or eGFP negative (eGFP-), depending on the reporter cassette used (AARP, AARB or AARG). Cells were grown in the presence of neomycin to eliminate cells that underwent spurious recombination before induction of DNA double-strand breaks (DSBs). DSBs were induced by transfection of an I-SceI endonuclease expression vector. (B) The reporter cassette was inserted using the Flp-In system (see S1 Fig, Materials and Methods) into a single genomic location in each cell line, which allows variants of the vector (i.e. different Alu elements) to be inserted in the same chromosomal location. (C) Repair of an I-SceI-induced DNA DSB may result in a deletion of the sequence between the two Alu elements after which the cell becomes neomycin sensitive (neos) and express the puromycin resistance (puroR), blasticidin resistance (blastR) or eGFP (eGFP+) gene. (D and E) I-SceI-induced DNA DSBs in the AARP (D) and AARB (E) HEK293FRT cells result in Alu/Alu recombination. Cells were transiently transfected with either I-SceI expression vector or empty vector (pUC19) control. Puromycin resistant (puror) or blasticidin resistant (blastr) colonies were counted and are shown graphically. Error bars denote standard error and statistical significance was shown using one-way ANOVA. Below the graphs are representative flasks showing puror or blastr colonies following I-SceI-induced DNA DSBs. (F) I-SceI-induced DNA DSBs in 0%-AARG HEK293FRT cells result in eGFP+ cells. The percentage of eGFP+ cells are shown graphically. Error bars represent standard error and statistical significance was shown using Student’s t-test. Representative flow plots from fluorescence activated cell sorting (FACS) analysis following transfections with I-SceI or empty vector pUC19 are shown below the graphs.

Mentions: We have designed a novel Alu/Alu recombination reporter cassette to quantitatively assess deletions caused by Alu elements. The basic construct consists of an human elongation factor 1α (EF1α) promoter upstream of two Alu sequences separated by approximately 1100 bp of sequence encoding a neomycin resistance (neoR) gene, a polyadenylation signal (pA), and an I-SceI endonuclease cleavage site to generate a DNA DSB between the two Alu elements (Fig. 1A). Maintaining the cells under neomycin selection eliminated almost all background Alu/Alu recombination that occurred prior to experimental treatment. A puromycin resistance (puroR) gene downstream of the Alu sequences is not initially expressed because it is separated from the EF1α promoter by the two Alu sequences, the neoR gene, and a polyadenylation signal (Fig. 1A and B). We designated this construct Alu/Alu recombination puromycin (AARP). If DNA repair results in a deletion of the sequence between the two Alu elements, the cells lose neoR, and the puroR gene comes into proximity of the EF1α promoter to confer puroR (Fig. 1C). Each component of the AARP reporter cassette is flanked with unique restriction enzyme sites to allow for rapid interchange of components in order to explore potential modulators of Alu/Alu recombination as well as different reporter genes (Fig. 1C and S1 Fig). In addition to recovering Alu/Alu recombination events, the AARP reporter system also can detect a subset of non-homologous end joining (NHEJ) events that result in ~1.0–1.9 kb deletions of the sequence near the I-SceI cleavage site but do not recreate a single Alu element (Fig. 2A). For simplicity, we will refer to these variable-length deletions as NHEJ events.


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 Alu/Alu recombination system.(A) Schematic of the Alu/Alu recombination reporter. Cells harboring the reporter were neomycin resistant (neor) and sensitive to puromycin (puros) or blasticidin (blasts) or eGFP negative (eGFP-), depending on the reporter cassette used (AARP, AARB or AARG). Cells were grown in the presence of neomycin to eliminate cells that underwent spurious recombination before induction of DNA double-strand breaks (DSBs). DSBs were induced by transfection of an I-SceI endonuclease expression vector. (B) The reporter cassette was inserted using the Flp-In system (see S1 Fig, Materials and Methods) into a single genomic location in each cell line, which allows variants of the vector (i.e. different Alu elements) to be inserted in the same chromosomal location. (C) Repair of an I-SceI-induced DNA DSB may result in a deletion of the sequence between the two Alu elements after which the cell becomes neomycin sensitive (neos) and express the puromycin resistance (puroR), blasticidin resistance (blastR) or eGFP (eGFP+) gene. (D and E) I-SceI-induced DNA DSBs in the AARP (D) and AARB (E) HEK293FRT cells result in Alu/Alu recombination. Cells were transiently transfected with either I-SceI expression vector or empty vector (pUC19) control. Puromycin resistant (puror) or blasticidin resistant (blastr) colonies were counted and are shown graphically. Error bars denote standard error and statistical significance was shown using one-way ANOVA. Below the graphs are representative flasks showing puror or blastr colonies following I-SceI-induced DNA DSBs. (F) I-SceI-induced DNA DSBs in 0%-AARG HEK293FRT cells result in eGFP+ cells. The percentage of eGFP+ cells are shown graphically. Error bars represent standard error and statistical significance was shown using Student’s t-test. Representative flow plots from fluorescence activated cell sorting (FACS) analysis following transfections with I-SceI or empty vector pUC19 are shown below the graphs.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005016.g001: The Alu/Alu recombination system.(A) Schematic of the Alu/Alu recombination reporter. Cells harboring the reporter were neomycin resistant (neor) and sensitive to puromycin (puros) or blasticidin (blasts) or eGFP negative (eGFP-), depending on the reporter cassette used (AARP, AARB or AARG). Cells were grown in the presence of neomycin to eliminate cells that underwent spurious recombination before induction of DNA double-strand breaks (DSBs). DSBs were induced by transfection of an I-SceI endonuclease expression vector. (B) The reporter cassette was inserted using the Flp-In system (see S1 Fig, Materials and Methods) into a single genomic location in each cell line, which allows variants of the vector (i.e. different Alu elements) to be inserted in the same chromosomal location. (C) Repair of an I-SceI-induced DNA DSB may result in a deletion of the sequence between the two Alu elements after which the cell becomes neomycin sensitive (neos) and express the puromycin resistance (puroR), blasticidin resistance (blastR) or eGFP (eGFP+) gene. (D and E) I-SceI-induced DNA DSBs in the AARP (D) and AARB (E) HEK293FRT cells result in Alu/Alu recombination. Cells were transiently transfected with either I-SceI expression vector or empty vector (pUC19) control. Puromycin resistant (puror) or blasticidin resistant (blastr) colonies were counted and are shown graphically. Error bars denote standard error and statistical significance was shown using one-way ANOVA. Below the graphs are representative flasks showing puror or blastr colonies following I-SceI-induced DNA DSBs. (F) I-SceI-induced DNA DSBs in 0%-AARG HEK293FRT cells result in eGFP+ cells. The percentage of eGFP+ cells are shown graphically. Error bars represent standard error and statistical significance was shown using Student’s t-test. Representative flow plots from fluorescence activated cell sorting (FACS) analysis following transfections with I-SceI or empty vector pUC19 are shown below the graphs.
Mentions: We have designed a novel Alu/Alu recombination reporter cassette to quantitatively assess deletions caused by Alu elements. The basic construct consists of an human elongation factor 1α (EF1α) promoter upstream of two Alu sequences separated by approximately 1100 bp of sequence encoding a neomycin resistance (neoR) gene, a polyadenylation signal (pA), and an I-SceI endonuclease cleavage site to generate a DNA DSB between the two Alu elements (Fig. 1A). Maintaining the cells under neomycin selection eliminated almost all background Alu/Alu recombination that occurred prior to experimental treatment. A puromycin resistance (puroR) gene downstream of the Alu sequences is not initially expressed because it is separated from the EF1α promoter by the two Alu sequences, the neoR gene, and a polyadenylation signal (Fig. 1A and B). We designated this construct Alu/Alu recombination puromycin (AARP). If DNA repair results in a deletion of the sequence between the two Alu elements, the cells lose neoR, and the puroR gene comes into proximity of the EF1α promoter to confer puroR (Fig. 1C). Each component of the AARP reporter cassette is flanked with unique restriction enzyme sites to allow for rapid interchange of components in order to explore potential modulators of Alu/Alu recombination as well as different reporter genes (Fig. 1C and S1 Fig). In addition to recovering Alu/Alu recombination events, the AARP reporter system also can detect a subset of non-homologous end joining (NHEJ) events that result in ~1.0–1.9 kb deletions of the sequence near the I-SceI cleavage site but do not recreate a single Alu element (Fig. 2A). For simplicity, we will refer to these variable-length deletions as NHEJ events.

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