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.
Highlights
DNA double-strand breaks (DSBs) are the most dangerous type of DNA damage due to their tendency to lead to chromosomal rearrangements, a hallmark of tumorigenesis, when they are repaired [1]
We use a new reporter assay to show that repair of DSBs results in Alumediated deletions that resolve through several distinct repair pathways
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
Summary
DNA double-strand breaks (DSBs) are the most dangerous type of DNA damage due to their tendency to lead to chromosomal rearrangements, a hallmark of tumorigenesis, when they are repaired [1]. One way in which chromosomal rearrangements occur in DSB repair is the use of non-allelic recombination between repetitive elements (reviewed in [2]), which comprise a large portion of the human genome [3]. The majority of identified Alu elements diverge 4%-20% from the consensus [3]. Despite this level of sequence divergence, Alu elements represent a major source of sequence homology in the human genome and contribute to genomic instability that arises from mutagenic recombination between these elements [4,5]. Alu/Alu recombination is estimated to cause as many as 0.5% of all new genetic diseases and is responsible for mutations that contribute to human cancers [4,5,6]
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