Yeast Tdp1 regulates the fidelity of nonhomologous end joining

Abstract

Tyrosyl-DNA-phosphodiesterase 1 (Tdp1) can disjoin peptides covalently bound to DNA. We assessed the role of Tdp1 in nonhomologous end joining (NHEJ) and found that linear DNA molecules with 5′ extensions showed a high frequency of misrepair in Δtdp1 cells. The joining errors in Δtdp1 cells were predominantly 2-4 nucleotide insertions. Ends with 3′ extensions or blunt ends did not show enhanced frequencies of errors, although Δtdp1 cells repaired blunt DNA ends with greater efficiency than WT cells. We found that insertions required Ku80 and DNA ligase IV, as well as polymerase IV. Our results show that yeast Tdp1 is a component of the NHEJ pathway. We suggest that Tdp1p 3′ nucleosidase activity regulates the processing of DNA ends by generating a 3′ phosphate, thereby restricting the ability of polymerases and other enzymes from acting at DNA ends. In support of this model, we found that overexpression of Tpp1, a yeast DNA 3′ phosphatase, also leads to a higher frequency of insertions, suggesting that the generation of a 3′ phosphate is a key step in Tdp1-mediated error prevention during NHEJ.Tpp1 | nucleosidase | repair accuracy | break repair N onhomologous end joining (NHEJ) is a critical pathway for repairing DNA double-strand breaks. In higher eukaryotes, it functions as a primary repair pathway for repairing doublestrand breaks from exogenous DNA damage and is also required for gene rearrangements in the immune system (1). This pathway is conserved in lower eukaryotes and bacteria where it functions mainly as a secondary repair pathway for the repair of doublestrand breaks (2, 3). Because NHEJ does not rely on DNA homology for carrying out repair, it is an intrinsically error-prone pathway. The detailed steps of how accuracy is maintained during NHEJ processes remain poorly understood.NHEJ is carried out by a set of well-conserved proteins including the Ku70/Ku80 proteins that bind to DNA ends, serving as a scaffold for subsequent repair reactions, and DNA ligase IV (4). In mammalian cells, a DNA-dependent protein kinase DNAPKcs also plays critical roles (5). In addition to the core factors that are absolutely required for the NHEJ pathway, other factors such as nucleases and DNA polymerases participate in NHEJ (6, 7).Tdp1p was identified on the basis of its ability to remove peptides covalently bound to DNA (8-10). Yeast Tdp1 can remove phosphotyrosyl-linked peptides from both the 3′ and 5′ ends of DNA (8,11). Tdp1 also has the ability to remove damaged and undamaged nucleosides from the 3′ end of DNA through an activity that is mechanistically identical to the esterase activity that removes peptides (12). Because human Tdp1 is mutated in a DNA repair disorder [spinocerebellar ataxia with axonal neuropathy (13)], we assessed additional roles of this enzyme by using yeast repair mutants. In this paper, we demonstrate that yeast Tdp1 plays an important and unique role in NHEJ and affects the accuracy of the formation of repair junctions. ResultsYeast Tdp1 is Required for Accurate Joining of some NHEJ Subst...

Frameshift Mutagenesis: The Roles of Primer–Template Misalignment and the Nonhomologous End-Joining Pathway inSaccharomyces cerevisiae

“…The tandem, 4-bp duplications seen here are of particular interest as they are similar to those recently reported using a plasmid-based NHEJ assay (Bahmed et al 2010(Bahmed et al , 2011. Because such duplications were observed only following transformation of linear molecules with cohesive 59 overhangs, it was proposed that they are generated by the precise ligation of filled-in, blunt ends ( Figure 5A).…”

“…Because such duplications were observed only following transformation of linear molecules with cohesive 59 overhangs, it was proposed that they are generated by the precise ligation of filled-in, blunt ends ( Figure 5A). In the plasmid-based assay, tandem duplications were elevated upon loss of either Tdp1 (Bahmed et al 2010) or Exo1 (Bahmed et al 2011). It was suggested that the 39-nucleosidase activity of Tdp1 converts the recessed 39-OH to a recessed 39-phosphate, thereby preventing the filling in of the enzyme-generated end (Bahmed et al 2010).…”

“…In the plasmid-based assay, tandem duplications were elevated upon loss of either Tdp1 (Bahmed et al 2010) or Exo1 (Bahmed et al 2011). It was suggested that the 39-nucleosidase activity of Tdp1 converts the recessed 39-OH to a recessed 39-phosphate, thereby preventing the filling in of the enzyme-generated end (Bahmed et al 2010). In the case of Exo1, either its 59 .…”

“…In a plasmid-based NHEJ assay, 4-bp duplications arise at a low frequency following transformation with linear molecules containing complementary, 4-nt 59 overhangs. Such events are specifically elevated in the absence of Tdp1, a 39 nucleosidase whose action presumably blocks the filling in of the recessed ends (Bahmed et al 2010). We thus examined whether loss of Tdp1 affects reversion of the lys2DA746,NR allele.…”

Repair of Double-Strand Breaks by End Joining

“…An interesting model developed in yeast proposes that Tdp1 removes intact bases from otherwise undamaged 5 0 -overhanging DSBs such that the new 3 0 -phosphate lesion prevents undesirable filling of the overhang and insertional mutagenesis (Fig. 4E) (Bahmed et al 2010). …”