Roles of base excision repair enzymes Nth1p and Apn2p from Schizosaccharomyces pombe in processing alkylation and oxidative DNA damage.
ABSTRACT Schizosaccharomyces pombe Nthpl, an ortholog of the endonuclease III family, is the sole bifunctional DNA glycosylase encoded in its genome. The enzyme removes oxidative pyrimidine and incises 3' to the apurinic/apyrimidinic (AP) site, leaving 3'-alpha,beta-unsaturated aldehyde. Analysis of nth1 cDNA revealed an intronless structure including 5'- and 3'-untranslated regions. An Nth1p-green fluorescent fusion protein was predominantly localized in the nuclei of yeast cells, indicating a nuclear function. Deletion of nth1 confirmed that Nth1p is responsible for the majority of activity for thymine glycol and AP site incision in the absence of metal ions, while nth1 mutants exhibit hypersensitivity to methylmethanesulfonate (MMS). Complementation of sensitivity by heterologous expression of various DNA glycosylases showed that the methyl-formamidopyrimidine (me-fapy) and/or AP sites are plausible substrates for Nth1p in repairing MMS damage. Apn2p, the major AP endonuclease in S. pombe, also greatly contributes to the repair of MMS damage. Deletion of nth1 from an apn2 mutant resulted in tolerance to MMS damage, indicating that Nth1p-induced 3'-blocks are responsible for MMS sensitivity in apn2 mutants. Overexpression of Apn2p in nth1 mutants failed to suppress MMS sensitivity. These results indicate that Nth1p, not Apn2p, primarily incises AP sites and that the resultant 3'-blocks are removed by the 3'-phosphodiesterase activity of Apn2p. Nth1p is dispensable for cell survival against low levels of oxidative stress, but wild-type yeast became more sensitive than the nth1 mutant at high levels. Overexpression of Nth1p in heavily damaged cells probably induced cell death via the formation of 3'-blocked single-strand breaks.
Article: Aprataxin, causative gene product for EAOH/AOA1, repairs DNA single-strand breaks with damaged 3'-phosphate and 3'-phosphoglycolate ends.[show abstract] [hide abstract]
ABSTRACT: Aprataxin is the causative gene product for early-onset ataxia with ocular motor apraxia and hypoalbuminemia/ataxia with oculomotor apraxia type 1 (EAOH/AOA1), the clinical symptoms of which are predominantly neurological. Although aprataxin has been suggested to be related to DNA single-strand break repair (SSBR), the physiological function of aprataxin remains to be elucidated. DNA single-strand breaks (SSBs) continually produced by endogenous reactive oxygen species or exogenous genotoxic agents, typically possess damaged 3'-ends including 3'-phosphate, 3'-phosphoglycolate, or 3'-alpha, beta-unsaturated aldehyde ends. These damaged 3'-ends should be restored to 3'-hydroxyl ends for subsequent repair processes. Here we demonstrate by in vitro assay that recombinant human aprataxin specifically removes 3'-phosphoglycolate and 3'-phosphate ends at DNA 3'-ends, but not 3'-alpha, beta-unsaturated aldehyde ends, and can act with DNA polymerase beta and DNA ligase III to repair SSBs with these damaged 3'-ends. Furthermore, disease-associated mutant forms of aprataxin lack this removal activity. The findings indicate that aprataxin has an important role in SSBR, that is, it removes blocking molecules from 3'-ends, and that the accumulation of unrepaired SSBs with damaged 3'-ends underlies the pathogenesis of EAOH/AOA1. The findings will provide new insight into the mechanism underlying degeneration and DNA repair in neurons.Nucleic Acids Research 02/2007; 35(11):3797-809. · 8.03 Impact Factor
Article: Early Steps in the DNA Base Excision Repair Pathway of a Fission Yeast Schizosaccharomyces pombe.[show abstract] [hide abstract]
ABSTRACT: DNA base excision repair (BER) accounts for maintaining genomic integrity by removing damaged bases that are generated endogenously or induced by genotoxic agents. In this paper, we describe the roles of enzymes functioning in the early steps of BER in fission yeast. Although BER is an evolutionarily conserved process, some unique features of the yeast repair pathway were revealed by genetic and biochemical approaches. AP sites generated by monofunctional DNA glycosylases are incised mainly by AP lyase activity of Nth1p, a sole bifunctional glycosylase in yeast, to leave a blocked 3' end. The major AP endonuclease Apn2p functions predominantly in removing the 3' block. Finally, a DNA polymerase fills the gap, and a DNA ligase seals the nick (Nth1p-dependent or short patch BER). Apn1p backs up Apn2p. In long patch BER, Rad2p endonuclease removes flap DNA containing a lesion after DNA synthesis. A UV-specific endonuclease Uve1p engages in an alternative pathway by nicking DNA on the 5' side of oxidative damage. Nucleotide excision repair and homologous recombination are involved in repair of BER intermediates including the AP site and single-strand break with the 3' block. Other enzymes working in 3' end processing are also discussed.Journal of nucleic acids 01/2010; 2010.
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ABSTRACT: Abasic (AP) sites are formed spontaneously and are inevitably intermediates during base excision repair of DNA base damages. AP sites are both mutagenic and cytotoxic and key enzymes for their removal are AP endonucleases. However, AP endonuclease independent repair initiated by DNA glycosylases performing β,δ-elimination cleavage of the AP sites has been described in mammalian cells. Here, we describe another AP endonuclease independent repair pathway for removal of AP sites in Schizosaccharomyces pombe that is initiated by a bifunctional DNA glycosylase, Nth1 and followed by cleavage of the baseless sugar residue by tyrosyl phosphodiesterase Tdp1. We propose that repair is completed by the action of a polynucleotide kinase, a DNA polymerase and finally a DNA ligase to seal the gap. A fission yeast double mutant of the major AP endonuclease Apn2 and Tdp1 shows synergistic increase in MMS sensitivity, substantiating that Apn2 and Tdp1 process the same substrate. These results add new knowledge to the complex cellular response to AP sites, which could be exploited in chemotherapy where synthetic lethality is a key strategy of treatment.Nucleic Acids Research 11/2011; 40(5):2000-9. · 8.03 Impact Factor