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.
BackgroundLINE-1 (L1) retrotransposons are common occupants of mammalian genomes representing about a fifth of the genetic content. Ongoing L1 retrotransposition in the germ line and somatic tissues has contributed to structural genomic variations and disease-causing mutations in the human genome. L1 mobilization relies on the function of two, self-encoded proteins, ORF1 and ORF2. The ORF2 protein contains two characterized domains: endonuclease and reverse transcriptase.ResultsUsing a bacterially purified endonuclease domain of the human L1 ORF2 protein, we have generated a monoclonal antibody specific to the human ORF2 protein. We determined that the epitope recognized by this monoclonal antibody includes amino acid 205, which is required for the function of the L1 ORF2 protein endonuclease. Using an in vitro L1 cleavage assay, we demonstrate that the monoclonal anti-ORF2 protein antibody partially inhibits L1 endonuclease activity without having any effect on the in vitro activity of the human AP endonuclease.ConclusionsOverall, our data demonstrate that this anti-ORF2 protein monoclonal antibody is a useful tool for human L1-related studies and that it provides a rationale for the development of antibody-based inhibitors of L1-induced damage.Electronic supplementary materialThe online version of this article (doi:10.1186/s13100-014-0029-x) contains supplementary material, which is available to authorized users.
Undergraduate research is a valuable experience that increases the likelihood of a STEM major to continue on to postgraduate training in their field. For students from groups underrepresented in the biomedical sciences, a strong mentoring relationship during this undergraduate period is a key component in preparing them for the next stage of their education and can have a significant influence on their ability to persist in the pipeline. Although the ideal scenario to increase the diversity of the biomedical workforce is to provide more BIPOC (Black, Indigenous, People of Color) faculty mentors for our undergraduates, we also need to develop strategies to provide strong mentoring experiences for our BIPOC students when those mentors are not in great number. At Xavier University of Louisiana, we have used our NIH BUILD Project Pathways program to look more closely at the mentor matching process. Throughout the past seven years, we have moved from the traditional mentor, research-focused matching process to a student-centered process. The lessons learned here can be used by any University looking to craft an inclusive undergraduate research program to meet the needs of all students, but in particular a diverse student population.
Background: LINE-1 (L1) retrotransposons are common occupants of mammalian genomes representing about a fifth of the genetic content. Ongoing L1 retrotransposition in the germ line and somatic tissues has contributed to structural genomic variations and disease-causing mutations in the human genome. L1 mobilization relies on the function of two, self-encoded proteins, ORF1 and ORF2. The ORF2 protein contains two characterized domains: endonuclease and reverse transcriptase. Results: Using a bacterially purified endonuclease domain of the human L1 ORF2 protein, we have generated a monoclonal antibody specific to the human ORF2 protein. We determined that the epitope recognized by this monoclonal antibody includes amino acid 205, which is required for the function of the L1 ORF2 protein endonuclease. Using an in vitro L1 cleavage assay, we demonstrate that the monoclonal anti-ORF2 protein antibody partially inhibits L1 endonuclease activity without having any effect on the in vitro activity of the human AP endonuclease.
Our lab studies the mammalian mobile long interspersed element 1 (LINE1). LINE1 elements mobilize in the genome through a copy and paste itself into new genomic loci through the process of retrotransposition. The full‐length LINE1 element is comprised of two open reading frames, ORF‐1 and ORF‐2. The latter protein, ORF‐2, encodes an endonuclease, which is responsible for nicking DNA near the adenine‐thymine rich base sequence. Proper functioning of LINE1 endonuclease facilitates the mobilization of the element and requires the formation of DNA damage in the form of double‐strand breaks (DSBs), which could, in turn, lead to diseases such as cancer. Approximately eighteen percent of the human genome is comprised of the LINE1 sequence and the expression of LINE1 proteins and mobilization of the LINE1 element has been implicated in several cancers, such as breast and prostate cancers. While events of LINE1 insertion may be mutagenic, the effect of the LINE1 endonuclease on genetic instability is unclear. If the DNA DSBs associated with LINE1 endonuclease are not repaired faithfully, then mutations may result that lead to disease onset or progression. In our laboratory we have identified small molecule inhibitors of the LINE1 endonuclease. Here we present our work investigating the impact of the small molecule inhibitors on the formation of LINE1 endonuclease associated DNA DSBs.Support or Funding InformationThe work presented is supported in part by monies from P20GM103424, TL4GM118968 and 5RL5GM118966‐03 as well as the Louisiana Cancer Research Consortium. We also thank the Molecular and Cellular Biology core at Xavier University of Louisiana.
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