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-Thr(540)
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.