Efficient Processing of DNA Ends during Yeast Nonhomologous End Joining

Abstract

Repair of DNA double strand breaks by nonhomolo- gous end joining (NHEJ) requires enzymatic processing beyond simple ligation when the terminal bases are damaged or not fully compatible. We transformed yeast with a series of linearized plasmids to examine the role of Pol4 (Pol IV, DNA polymerase) in repair at a variety of end configurations. Mutation of POL4 did not impair DNA polymerase-independent religation of fully com- patible ends and led to at most a 2-fold reduction in the frequency of joins that require only DNA polymeriza- tion. In contrast, the frequency of joins that also re- quired removal of a 5-or3-terminal mismatch was markedly reduced in pol4 (but not rev3, exo1, apn1,or rad1) yeast. In a chromosomal double strand break as- say, pol4 mutation conferred a marked increase in sen- sitivity to HO endonuclease in a rad52 background, due primarily to loss of an NHEJ event that anneals with a 3-terminal mismatch. The NHEJ activity of Pol4 was dependent on its nucleotidyl transferase function, as well as its unique amino terminus. Paradoxically, in vitro analyses with oligonucleotide substrates demon- strated that although Pol4 fills gaps with displacement of mismatched but not matched 5 termini, it lacks both 5- and 3-terminal nuclease activities. Pol4 is thus spe- cifically recruited to perform gap-filling in an NHEJ pathway that must also involve as yet unidentified nucleases.