Transcription of a Donor Enhances Its Use during Double-Strand Break-Induced Gene Conversion in Human Cells

Abstract

Homologous recombination (HR) mediates accurate repair of double-strand breaks (DSBs) but carries the risk of large-scale genetic change, including loss of heterozygosity, deletions, inversions, and translocations. Nearly one-third of the human genome consists of repetitive sequences, and DSB repair by HR often requires choices among several homologous repair templates, including homologous chromosomes, sister chromatids, and linked or unlinked repeats. Donor preference during DSB-induced gene conversion was analyzed by using several HR substrates with three copies of neo targeted to a human chromosome. Repair of I-SceI nucleaseinduced DSBs in one neo (the recipient) required a choice between two donor neo genes. When both donors were downstream, there was no significant bias for proximal or distal donors. When donors flanked the recipient, we observed a marked (85%) preference for the downstream donor. Reversing the HR substrate in the chromosome eliminated this preference, indicating that donor choice is influenced by factors extrinsic to the HR substrate. Prior indirect evidence suggested that transcription might increase donor use. We tested this question directly and found that increased transcription of a donor enhances its use during gene conversion. A preference for transcribed donors would minimize the use of nontranscribed (i.e., pseudogene) templates during repair and thus help maintain genome stability.DNA double-strand breaks (DSBs) are potentially lethal events that can be repaired by homologous recombination (HR) or nonhomologous end-joining (NHEJ). If left unrepaired, DSBs can lead to chromosome loss or cell death. DSBs are caused by ionizing radiation, X-rays, free radicals, chemicals, and nucleases and may occur at stalled replication forks (30). Although DSB repair can be accurate, misrepair can have serious consequences. Genomic rearrangements arising during DSB repair can lead to loss of heterozygosity and, ultimately, carcinogenesis through the activation of proto-oncogenes or inactivation of tumor suppressor genes (4, 45). HR appears to be essential for maintaining genome stability. Cells with defects in HR proteins such as BRCA1/2, XRCC2/3, and other RAD51 paralogs exhibit high levels of genomic instability (6,18,28,42,43,52,68). DSBs are a critical type of DNA damage; unlike single-strand breaks, which have a readily available template for repair, DSB repair by HR requires a search for a homologous template.Repetitive elements make up one-third of the mammalian genome and consist of coding DNA, and noncoding DNA such as satellites that can exist in thousands of copies. Repetitive sequences are scattered throughout the genome, including SINE and LINE elements, and ribosomal RNA gene repeats. HR between linked or unlinked repetitive elements can result in a variety of rearrangements including translocations, duplications, and deletions (45).When DNA is broken, there are many potential homologous sequences that could be used as a repair template, including linked or unlinked repeats, sister...

Genome fractionation and loss of heterozygosity in hybrids and polyploids: mechanisms, consequences for selection, and link to gene function

“…Namely, LOH-positive genes have above-average expression levels, which is consistent with the hypothesis that DNA of transcriptionally active loci is more relaxed and, hence, prone to double strand breaks (DSB), followed by repair cascade, including the recombination machinery (González-Barrera et al 2002;Cummings et al 2007). Cobitis hybrids also tend to replace the less expressed parental allele by the more expressed allele, which is in-line with growing evidence that more transcribed homoeologs are preferably utilized as templates during DSB-induced gene conversion (Schildkraut et al 2006). Finally, the prevalence of AT → GC substitutions on some LOH sites conforms to the expected GC bias in template preference (Duret and Galtier 2009;Williams et al 2015).…”

“…We may also speculate that biased retention of the C. elongatoides subgenome may reflect its special "mechanistic" properties. Several reasons for biased template preference have already been proposed, including different expression levels of orthologous alleles (Schildkraut et al 2006), different GC contents (Duret and Galtier 2009;Williams et al 2015), or the effect of maternal ancestry when maternal endonuclease systems may preferentially induce DSB on paternal chromosomes, thereby causing biased DSB repair and unequal gene conversion (Wang et al 2010). However, none of these hypotheses may sufficiently explain the prevalence of E-like LOH as the C. elongatoides subgenome possesses neither higher expression levels nor different GC content and the maternal ancestor of all studied elongatoides-taenia hybrids was C. taenia.…”

Transcription as a source of genome instability

“…Although most of the stimulatory effects of transcription on homologous recombination likely come through the generation of recombination-initiating lesions 116 , this is not the entire story. Transcription additionally seems to enhance access to the repair template in both budding yeast and human cells 117,118 . In addition to the process of transcription, the primary transcript itself may be relevant to recombination processes.…”

Parallel Concerted Evolution of Ribosomal Protein Genes in Fungi and Its Adaptive Significance

“…Through gene conversion, the sequences of paralogous genes are homogenized, easing the new mutations accumulated in one of the paralogous genes. It has been shown that highly-expressed genes are more likely to experience mRNA-mediated gene conversion (Weng, et al 2000; Schildkraut, et al 2006). Thus, as a group of most actively transcribed genes (Warner 1999), the repeated occurrence of gene conversion on RP genes might be due to the highly expressed nature of RP genes.…”