A role for FEN-1 in nonhomologous DNA end joining: The order of strand annealing and nucleolytic processing events

Eukaryotic repair of double-strand DNA breaks can occur either by homologou s recombination or by nonhomologous DNA end joining (NHEJ). NHEJ relies on Ku70/86, XRCC4, DNA ligase IV, and DNA-dependent protein kinase. NHEJ invol ves a synapsis step in which the two ends are maintained in proximity, proc essing steps in which nucleases and polymerases act on the ends, an alignme nt step in which a few nucleotides of terminal homology guide the ends into preferred alignments, and a ligation step. Some of the steps, such as liga tion, rely on a single enzymatic component. However, the processing steps b egin and end with a wide array of alternative substrates and products, resp ectively, and likely involve multiple nucleases and polymerases. Given the alternative pathways that can be catalyzed by the remaining nucleases and p olymerases, no one of these processing enzymes is likely to be essential, T he only requirement for the processing enzymes, as a collective, is to gene rate a ligatable configuration, namely a ligatable nick on each strand. Her e, we have tested the two major known 5'-specific nucleases of Saccharomyce s cerevisiae for involvement in NHEJ. Whereas EXO1 does not appear to be in volved to any detectable level, deleting RAD27 (FEN-1 of yeast) leads to a 4.4-fold reduction specifically of those NHEJ events predicted to proceed b y means of 5' flap intermediates, Because Rad27/FEN-1 acts specifically at 5' flap structures, these results suggest that the NHEJ alignment step prec edes nucleolytic processing steps in a significant fraction of NHEJ events.