Abstract
DNA double-strand breaks (DSBs) are one of the most dangerous DNA lesions, since their erroneous repair by nonhomologous end-joining (NHEJ) can generate harmful chromosomal rearrangements. PolX DNA polymerases are well suited to extend DSB ends that cannot be directly ligated due to their particular ability to bind to and insert nucleotides at the imperfect template-primer structures formed during NHEJ. Herein, we have devised genetic assays in yeast to induce simultaneous DSBs in different chromosomes in vivo. The repair of these breaks in trans could result in reciprocal chromosomal translocations that were dependent on classical Ku-dependent NHEJ. End-joining events leading to translocations were mainly based on the formation of short base pairing between 3′-overhanging DNA ends coupled to gap-filling DNA synthesis. A major proportion of these events were specifically dependent on yeast DNA polymerase Pol4 activity. In addition, we have discovered that Pol4-Thr540 amino acid residue can be phosphorylated by Tel1/ATM kinase, which could modulate Pol4 activity during NHEJ. Our data suggest that the role of Tel1 in preventing break-induced chromosomal translocations can, to some extent, be due to its stimulating effect on gap-filling activity of Pol4 to repair DSBs in cis. Overall, this work provides further insight to the molecular mechanisms of DSB repair by NHEJ and presents a new perspective to the understanding of how chromosomal translocations are formed in eukaryotic cells.
Highlights
DNA double-strand breaks (DSBs) are one of the most cytotoxic lesions
They are often generated from DNA double-strand breaks that are inaccurately repaired by DNA repair machinery
We found evidence showing that the classical nonhomologous end-joining repair pathway can be a source of chromosomal translocations, with a relevant role for yeast DNA polymerase Pol4 in such processes
Summary
DNA double-strand breaks (DSBs) are one of the most cytotoxic lesions. They can originate during cellular metabolism or upon exposure to DNA damaging agents such as radiation or chemicals. Despite the ability of NHEJ to join breaks directly, most DSBs occurring in vivo are not fully complementary or have chemical modifications at their ends, and cannot be directly ligated. In these cases, additional processing, such as DNA end trimming or gap-filling DNA synthesis, may be required in order to optimize base pairing before ligaton [7]. The Ku complex initially mediates the synapsis between the two broken DNA ends, protecting them from extensive degradation. Thereafter, it recruits other components, such as the XRCC4/DNA Ligase IV complex. In the absence of Ku, or due to its departure from DSB ends, the occurrence of alt-NHEJ increases relative to the extent of DSB resection, as it allows uncovering larger microhomologies to be used for end-joining [9]
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