title: The long N-terminus of the C. elegans DNA repair enzyme APN-1 targets the protein to the nucleus of a heterologous system.
abstract: We previously isolated from a Caenorhabditis elegans cDNA library, designed for two-hybrid screening, a gene encoding the DNA repair enzyme APN-1 using cross-specie complementation analysis of the Saccharomyces cerevisiae apn1∆ apn2∆ tpp1∆ triple mutant deficient in the ability to repair several types of DNA lesions including apurinic/apyrimidinic (AP) sites. We subsequently purified the APN-1 from this yeast mutant and demonstrated that it possesses four distinct DNA repair activities. However, following the re-annotation of the C. elegans genome we discovered that the functionally active APN-1 encoded by the cDNA from the library might lack 108 amino acid residues from the N-terminus. We therefore synthesized the entire C. elegans apn-1 gene encoding the putative full-length APN-1 and created several N-terminal deletion mutants lacking either 63, 83 or 118 amino acid residues. The full-length APN-1, APN-1 (1-63Δ) and APN-1 (1-83Δ), but not APN-1 (1-118Δ) were stably expressed in the yeast triple mutant and cleaved the AP site substrate. However, only the full-length APN-1 rescued the yeast mutant from the genotoxicity caused by methyl methane sulfonate, a DNA damaging agent that creates AP sites in the genome. The full-length APN-1 was localized to the yeast nucleus, while APN-1 (1-63Δ) and APN-1 (1-83Δ) retained a cytoplasmic distribution. Our data suggest that the N-terminal region has no direct role in the DNA repair functions of APN-1 other than to target the protein to the nucleus and possibly to maintain its stability. Thus, the truncated APN-1, previously isolated from the two-hybrid library, ability to complement the yeast triple mutant depends on the engineered SV40 nuclear localization signal.
authors.affiliation: Maisonneuve-Rosemont Hospital, Research Center, Université de Montréal, 5415 Boul. de l' Assomption, Montréal, Québec H1T 2M4, Canada.
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Maisonneuve-Rosemont Hospital, Research Center, Université de Montréal, 5415 Boul. de l' Assomption, Montréal, Québec H1T 2M4, Canada.
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Yang
authors.affiliation: Maisonneuve-Rosemont Hospital, Research Center, Université de Montréal, 5415 Boul. de l' Assomption, Montréal, Québec H1T 2M4, Canada.
authors.forename: Abdelghani
authors.initials: A
authors.lastname: Mazouzi
authors.affiliation: Maisonneuve-Rosemont Hospital, Research Center, Université de Montréal, 5415 Boul. de l' Assomption, Montréal, Québec H1T 2M4, Canada. Electronic address: dindial.ramotar@umontreal.ca.
authors.forename: Dindial
authors.initials: D
authors.lastname: Ramotar
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000201:enzymology*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000378:metabolism*; D002467:Cell Nucleus / Q000201:enzymology*; D017931:DNA Primers; D004260:DNA Repair; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid
chemical_list: D029742:Caenorhabditis elegans Proteins; D017931:DNA Primers; D004706:Endodeoxyribonucleases; C102951:apn-1 protein, C elegans
keywords: AP endonuclease; C. elegans; Cross-specie complementation; DNA repair; Nuclear localization signal; Yeast