Intrinsic 5'-deoxyribose-5-phosphate lyase activity in Saccharomyces cerevisiae Trf4 protein with a possible role in base excision DNA repair.
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 beta (Polbeta). 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 Polbeta. Previous studies of yeast trf4Delta mutants revealed hypersensitivity to methylmethane sulfonate (MMS) but not UV light, a characteristic of BER mutants in other organisms. We found that, like mammalian Polbeta, 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 Polbeta, 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 trf4Delta mutation in a rad27Delta background, in an AP lyase-deficient background (ogg1 ntg1 ntg2), or in a pol4Delta 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 rad27Delta 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.
Gellon
Lionel
L
Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, MA 02115, USA.
Carson
Dena R
DR
Carson
Jonathan P
JP
Demple
Bruce
B
D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D045585:Flap Endonucleases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D009841:Oligonucleotides / Q000235:genetics; D012267:Ribosemonophosphates / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
C502791:5'-deoxyribose-5-phosphate; D009841:Oligonucleotides; D012267:Ribosemonophosphates; D029701:Saccharomyces cerevisiae Proteins; D012321:DNA-Directed RNA Polymerases; C506067:Trf5 protein, S cerevisiae; D004259:DNA-Directed DNA Polymerase; C487545:PAP2 protein, S cerevisiae; D045585:Flap Endonucleases; C468323:RAD27 protein, S cerevisiae