Intrinsic 5′-deoxyribose-5-phosphate lyase activity in Saccharomyces cerevisiae Trf4 protein with a possible role in base excision DNA repair

Abstract

In Saccharomyces cerevisiae, the base excision DNA repair (BER) pathway has been thought to involve only a multinucleotide (long-patch) mechanism (LP-BER), in contrast to most known cases that include a major single-nucleotide pathway (SN-BER). The key step in mammalian SN-BER, removal of the 5′-terminal abasic residue generated by AP endonuclease incision, is effected by DNA polymerase β (Polβ). Computational analysis indicates that yeast Trf4 protein, with roles in sister chromatin cohesion and RNA quality control, is a new member of the X family of DNA polymerases that includes Polβ. Previous studies of yeast trf4Δ mutants revealed hypersensitivity to methylmethane sulfonate (MMS) but not UV light, a characteristic of BER mutants in other organisms. We found that, like mammalian Polβ, Trf4 is able to form a Schiff base intermediate with a 5′-deoxyribose-5-phosphate substrate and to excise the abasic residue through a dRP lyase activity. Also like Polβ, Trf4 forms stable cross-links in vitro to 5′-incised 2-deoxyribonolactone residues in DNA. We determined the sensitivity to MMS of strains with a trf4Δ mutation in a rad27Δ background, in an AP lyase-deficient background ( ogg1 ntg1 ntg2), or in a pol4Δ background. Only a RAD27 genetic interaction was detected: there was higher sensitivity for strains mutated in both TRF4 and RAD27 than either single mutant, and overexpression of Trf4 in a rad27Δ background partially suppressed MMS sensitivity. The data strongly suggest a role for Trf4 in a pathway parallel to the Rad27-dependent LP-BER in yeast. Finally, we demonstrate that Trf5 significantly affects MMS sensitivity and thus probably BER efficiency in cells expressing either wild-type Trf4 or a C-terminus-deleted form.