Abstract
Homologous recombination (HR) is essential for accurate genome duplication and maintenance of genome stability. In eukaryotes, chromosomal double strand breaks (DSBs) are central to HR during specialized developmental programs of meiosis and antigen receptor gene rearrangements, and form at unusual DNA structures and stalled replication forks. DSBs also result from exposure to ionizing radiation, reactive oxygen species, some anti-cancer agents, or inhibitors of topoisomerase II. Literature predicts that repair of such breaks normally will occur by non-homologous end-joining (in G1), intrachromosomal HR (all phases), or sister chromatid HR (in S/G2). However, no in vivo model is in place to directly determine the potential for DSB repair in somatic cells of mammals to occur by HR between repeated sequences on heterologs (i.e., interchromosomal HR). To test this, we developed a mouse model with three transgenes—two nonfunctional green fluorescent protein (GFP) transgenes each containing a recognition site for the I-SceI endonuclease, and a tetracycline-inducible I-SceI endonuclease transgene. If interchromosomal HR can be utilized for DSB repair in somatic cells, then I-SceI expression and induction of DSBs within the GFP reporters may result in a functional GFP+ gene. Strikingly, GFP+ recombinant cells were observed in multiple organs with highest numbers in thymus, kidney, and lung. Additionally, bone marrow cultures demonstrated interchromosomal HR within multiple hematopoietic subpopulations including multi-lineage colony forming unit–granulocyte-erythrocyte-monocyte-megakaryocte (CFU-GEMM) colonies. This is a direct demonstration that somatic cells in vivo search genome-wide for homologous sequences suitable for DSB repair, and this type of repair can occur within early developmental populations capable of multi-lineage differentiation.
Highlights
Faithful repair of DNA damage, including double-strand breaks (DSBs), is crucial to genome stability and normal cell survival and proliferation [1]
Studies in multiple organisms have demonstrated that EJ is most efficient in G1 and in noncycling somatic cells while homology-directed DSB repair is favored in both S/G2 utilizing a sister chromatid and intrachromosomal homologous recombination (HR) [19,21,22,23,24,25,26]
Homologs are utilized for HR-directed DSB repair with lower efficiency this is increased in organisms that exhibit a high degree of mitotic pairing, supporting the hypothesis that proximity of homologous sequences is an important factor in determining template choice [31,32,33]
Summary
Faithful repair of DNA damage, including double-strand breaks (DSBs), is crucial to genome stability and normal cell survival and proliferation [1]. DSBs are potent inducers of recombination and increase both homologous recombination (HR) and non-homologous end-joining (EJ) events by several orders of magnitude [19,20]. These two major DSB repair pathways differ based on their requirement for a donor DNA template with significant sequence homology; their relative activity changes with each stage of the cell cycle. Studies in multiple organisms have demonstrated that EJ is most efficient in G1 and in noncycling somatic cells while homology-directed DSB repair is favored in both S/G2 utilizing a sister chromatid and intrachromosomal HR [19,21,22,23,24,25,26]. Whether repair of DSBs in vivo in mammals occurs by interchromosomal HR at significant and detectable frequencies has not been demonstrated
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
