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1.  The Contribution of Alu Elements to Mutagenic DNA Double-Strand Break Repair 
PLoS Genetics  2015;11(3):e1005016.
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.
Author Summary
DNA double-strand breaks (DSBs) are a highly mutagenic form of DNA damage that can be repaired through one of several pathways with varied degrees of sequence preservation. Faithful repair of DSBs often occurs through gene conversion in which a sister chromatid is used as a repair template. Unfaithful repair of DSBs can occur through non-allelic homologous or homeologous recombination, which leads to chromosomal abnormalities such as deletions, duplications, and translocations and has been shown to cause several human genetic diseases. Substrates for these homologous and homeologous events include Alu elements, which are approximately 300 bp elements that comprise ~11% of the human genome. We use a new reporter assay to show that repair of DSBs results in Alu-mediated deletions that resolve through several distinct repair pathways. Either single-strand annealing (SSA) repair or microhomology-mediated end joining occurs ‘in register’ between two Alu elements when Alu sequence divergence is low. However, with more diverged Alu elements, like those typically found in the human genome, repair of DSBs appears to use the Alu/Alu homeology to direct non-homologous end joining in the general vicinity of the Alu elements. Mutagenic NHEJ repair involving divergent Alu elements may represent a common repair event in primate genomes.
PMCID: PMC4356517  PMID: 25761216
2.  A droplet digital PCR detection method for rare L1 insertions in tumors 
Mobile DNA  2014;5:30.
The active human mobile element, long interspersed element 1 (L1) currently populates human genomes in excess of 500,000 copies per haploid genome. Through its mobility via a process called target primed reverse transcription (TPRT), L1 mobilization has resulted in over 100 de novo cases of human disease and has recently been associated with various cancer types. Large advances in high-throughput sequencing (HTS) technology have allowed for an increased understanding of the role of L1 in human cancer; however, researchers are still limited by the ability to validate potentially rare L1 insertion events detected by HTS that may occur in only a small fraction of tumor cells. Additionally, HTS detection of rare events varies greatly as a function of read depth, and new tools for de novo element discovery are needed to fill in gaps created by HTS.
We have employed droplet digital PCR (ddPCR) to detect rare L1 loci in mosaic human genomes. Our assay allows for the detection of L1 insertions as rare as one cell in every 10,000.
ddPCR represents a robust method to be used alongside HTS techniques for detecting, validating and quantitating rare L1 insertion events in tumors and other tissues.
Electronic supplementary material
The online version of this article (doi:10.1186/s13100-014-0030-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4297411  PMID: 25598847
L1; retrotransposon; droplet digital PCR; tumor
3.  Driving Scenes test of the Neuropsychological Assessment Battery (NAB) and on-road driving performance in aging and very mild dementia 
The Driving Scenes test of the new Neuropsychological Assessment Battery (NAB; [Stern, R.A., & White, T. (2003a). Neuropsychological Assessment Battery. Lutz, FL: Psychological Assessment Resources, Inc.]) measures several aspects of visual attention thought to be important for driving ability. The current study examined the relationship between scores on the Driving Scenes test and on-road driving performance on a standardized driving test. Healthy participants performed significantly better on the Driving Scenes test than did very mildly demented participants. A correlation of 0.55 was found between the brief, office-based Driving Scenes test and the 108-point on-road driving score. Furthermore, the Driving Scenes test scores differed significantly across the driving instructor’s three global ratings (safe, marginal, and unsafe), and results of a discriminant function analysis indicated that the Driving Scenes test correctly classified 66% of participants into these groups. Thus, the new NAB Driving Scenes test appears to have good ecological validity for real-world driving ability in normal and very mildly demented older adults.
PMCID: PMC3292213  PMID: 15708731
Driving; Aging; Dementia; Neuropsychology; Attention; Visual
4.  The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4–Dependent and –Independent Mechanisms 
PLoS Genetics  2012;8(2):e1002534.
Mobile group II introns are bacterial retrotransposons that are thought to have invaded early eukaryotes and evolved into introns and retroelements in higher organisms. In bacteria, group II introns typically retrohome via full reverse splicing of an excised intron lariat RNA into a DNA site, where it is reverse transcribed by the intron-encoded protein. Recently, we showed that linear group II intron RNAs, which can result from hydrolytic splicing or debranching of lariat RNAs, can retrohome in eukaryotes by performing only the first step of reverse splicing, ligating their 3′ end to the downstream DNA exon. Reverse transcription then yields an intron cDNA, whose free end is linked to the upstream DNA exon by an error-prone process that yields junctions similar to those formed by non-homologous end joining (NHEJ). Here, by using Drosophila melanogaster NHEJ mutants, we show that linear intron RNA retrohoming occurs by major Lig4-dependent and minor Lig4-independent mechanisms, which appear to be related to classical and alternate NHEJ, respectively. The DNA repair polymerase θ plays a crucial role in both pathways. Surprisingly, however, mutations in Ku70, which functions in capping chromosome ends during NHEJ, have only moderate, possibly indirect effects, suggesting that both Lig4 and the alternate end-joining ligase act in some retrohoming events independently of Ku. Another potential Lig4-independent mechanism, reverse transcriptase template switching from the intron RNA to the upstream exon DNA, occurs in vitro, but gives junctions differing from the majority in vivo. Our results show that group II introns can utilize cellular NHEJ enzymes for retromobility in higher organisms, possibly exploiting mechanisms that contribute to retrotransposition and mitigate DNA damage by resident retrotransposons. Additionally, our results reveal novel activities of group II intron reverse transcriptases, with implications for retrohoming mechanisms and potential biotechnological applications.
Author Summary
Group II introns are bacterial mobile elements thought to be ancestors of introns and retrotransposons in higher organisms. They consist of a catalytically active intron RNA and an intron-encoded reverse transcriptase, which function together to promote intron integration into new DNA sites in a process called “retrohoming.” In bacteria, retrohoming occurs by the excised intron lariat RNA fully reverse splicing into a DNA site, where it is reverse transcribed, yielding an intron cDNA that is copied directly into the host genome. However, little is known about how group II introns behave in higher organisms. Here, we find that linear group II intron RNAs, which cannot fully reverse splice, retrohome in Drosophila melanogaster by attaching themselves to only one end of a DNA site. Reverse transcription then yields an intron cDNA, which is integrated into the recipient DNA by host enzymes that function in non-homologous end joining, a critical cellular DNA–repair pathway. Biochemical experiments exploring alternate mechanisms show that group II intron reverse transcriptases can also template switch efficiently from one RNA template to a second RNA or DNA template, thereby directly linking the two template sequences. Our findings have implications for retotransposition and DNA repair mechanisms and potential biotechnological applications.
PMCID: PMC3280974  PMID: 22359518
5.  Group II Intron-Based Gene Targeting Reactions in Eukaryotes 
PLoS ONE  2008;3(9):e3121.
Mobile group II introns insert site-specifically into DNA target sites by a mechanism termed retrohoming in which the excised intron RNA reverse splices into a DNA strand and is reverse transcribed by the intron-encoded protein. Retrohoming is mediated by a ribonucleoprotein particle that contains the intron-encoded protein and excised intron RNA, with target specificity determined largely by base pairing of the intron RNA to the DNA target sequence. This feature enabled the development of mobile group II introns into bacterial gene targeting vectors (“targetrons”) with programmable target specificity. Thus far, however, efficient group II intron-based gene targeting reactions have not been demonstrated in eukaryotes.
Methodology/Principal Findings
By using a plasmid-based Xenopus laevis oocyte microinjection assay, we show that group II intron RNPs can integrate efficiently into target DNAs in a eukaryotic nucleus, but the reaction is limited by low Mg2+ concentrations. By supplying additional Mg2+, site-specific integration occurs in up to 38% of plasmid target sites. The integration products isolated from X. laevis nuclei are sensitive to restriction enzymes specific for double-stranded DNA, indicating second-strand synthesis via host enzymes. We also show that group II intron RNPs containing either lariat or linear intron RNA can introduce a double-strand break into a plasmid target site, thereby stimulating homologous recombination with a co-transformed DNA fragment at frequencies up to 4.8% of target sites. Chromatinization of the target DNA inhibits both types of targeting reactions, presumably by impeding RNP access. However, by using similar RNP microinjection methods, we show efficient Mg2+-dependent group II intron integration into plasmid target sites in zebrafish (Danio rerio) embryos and into plasmid and chromosomal target sites in Drosophila melanogster embryos, indicating that DNA replication can mitigate effects of chromatinization.
Our results provide an experimental foundation for the development of group II intron-based gene targeting methods for higher organisms.
PMCID: PMC2518211  PMID: 18769669

Results 1-5 (5)