Abstract
Polymerase theta-mediated end joining (TMEJ) is a chromosome break repair pathway that is able to rescue the lethality associated with the loss of proteins involved in early steps in homologous recombination (e.g., BRCA1/2). This is due to the ability of polymerase theta (Pol θ) to use resected, 3' single stranded DNA tails to repair chromosome breaks. These resected DNA tails are also the starting substrate for homologous recombination. However, it remains unknown if TMEJ can compensate for the loss of proteins involved in more downstream steps during homologous recombination. Here we show that the Holliday junction resolvases SLX4 and GEN1 are required for viability in the absence of Pol θ in Drosophila melanogaster, and lack of all three proteins results in high levels of apoptosis. Flies deficient in Pol θ and SLX4 are extremely sensitive to DNA damaging agents, and mammalian cells require either Pol θ or SLX4 to survive. Our results suggest that TMEJ and Holliday junction formation/resolution share a common DNA substrate, likely a homologous recombination intermediate, that when left unrepaired leads to cell death. One major consequence of Holliday junction resolution by SLX4 and GEN1 is cancer-causing loss of heterozygosity due to mitotic crossing over. We measured mitotic crossovers in flies after a Cas9-induced chromosome break, and observed that this mutagenic form of repair is increased in the absence of Pol θ. This demonstrates that TMEJ can function upstream of the Holiday junction resolvases to protect cells from loss of heterozygosity. Our work argues that Pol θ can thus compensate for the loss of the Holliday junction resolvases by using homologous recombination intermediates, suppressing mitotic crossing over and preserving the genomic stability of cells.
Highlights
Double-strand breaks (DSBs) are a toxic form of DNA damage
Mutations in genes involved in the early steps of homologous recombination (BRCA1 and BRCA2), a mostly error-free chromosome break repair pathway, lead to hereditary breast cancer
Pol θ is required in the absence of factors that promote both non-homologous end joining (NHEJ) (e.g., KU70 and 53BP1) [12,13] and homologous recombination (HR) (e.g., BRCA1 and BRCA2) [13,14,15], showing that theta-mediated end joining (TMEJ) can compensate for their loss
Summary
Double-strand breaks (DSBs) are a toxic form of DNA damage. DSBs are generated during common cellular processes (e.g., replication, transcription), after exposure to ionizing radiation, or by specialized mechanisms such as meiosis or the development of the adaptive immune system [1]. Pol θ is required in the absence of factors that promote both NHEJ (e.g., KU70 and 53BP1) [12,13] and HR (e.g., BRCA1 and BRCA2) [13,14,15], showing that TMEJ can compensate for their loss. This is of particular interest in the context of HR-deficient breast and ovarian cancer, where Pol θ has been proposed as a promising therapeutic target [16]
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