Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins.

AP endonuclease; Leishmania amazonensis; lamap; Base Excision Repair; DNA repair

Authorship

Erika Verissimo-Villela

Universidade do Estado do Rio de Janeiro, Faculdade de Ciências Médicas, Departamento de Microbiologia, Imunologia e Parasitologia, Rio de Janeiro, RJ, BrasilUniversidade do Estado do Rio de JaneiroBrasilRio de Janeiro, RJ, BrasilUniversidade do Estado do Rio de Janeiro, Faculdade de Ciências Médicas, Departamento de Microbiologia, Imunologia e Parasitologia, Rio de Janeiro, RJ, Brasil

Milene Yoko Kitahara-Oliveira

Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório Interdisciplinar de Pesquisas Médicas, Rio de Janeiro, RJ, BrasilFundação Oswaldo CruzBrasilRio de Janeiro, RJ, BrasilFundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório Interdisciplinar de Pesquisas Médicas, Rio de Janeiro, RJ, Brasil

Ana Beatriz de Bragança dos Reis

Rodolpho Mattos Albano

Universidade do Estado do Rio de Janeiro, Instituto de Biologia Roberto Alcântara Gomes, Laboratório de Genoma, Rio de Janeiro, RJ, BrasilUniversidade do Estado do Rio de JaneiroBrasilRio de Janeiro, RJ, BrasilUniversidade do Estado do Rio de Janeiro, Instituto de Biologia Roberto Alcântara Gomes, Laboratório de Genoma, Rio de Janeiro, RJ, Brasil

Alda Maria Da-Cruz

Alexandre Ribeiro Bello ⁺+ Corresponding author: bello@uerj.br

+ Corresponding author: bello@uerj.br

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Figures

Figures (2)

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Fig. 1
: LAMAP harbors conserved region domains of the AP endonuclease superfamily. (A) Multiple amino acid (aa) sequences alignment of members of the AP endonuclease superfamily from Escherichia coli and human (AP1) and corresponding sequences found in trypanosomatids after BLAST using the Leishmania amazonensis putative AP endonuclease (LAMAP) sequence as template. Identical amino acids are highlighted in black and similar in gray. The main residue block of aa presented in all organisms aligned (LQETK) is highlighted in red. Asterisks indicate residues responsible for nuclear localisation signals. (B) Phylogenetic tree constructed by neighbor-joining of exonucleases/endonucleases/phosphatases superfamily aa sequences. ENDO III: Endonuclease III. ENDO IV: Endonuclease IV. EXO III: Exonuclease III. Human AP1: human AP Endonuclease 1. Bootstrap values are displayed on each tree branch.

Thumbnail

Fig. 2
: LAMAP restores DNA repair activity in Escherichia coli Base Excision Repair deficient strains. (A) E. coli wild type strain AB1157 (gray column) triple null mutant BW535 (white column) and triple mutant transformed with pLamap (black column) challenged with increasing concentrations of menadione. (B) E. coli triple null mutant BW535 (gray column) and triple mutant transformed with pLamap (black column) challenged with increasing concentrations of aminofluorene. ***p > 0.001; **p > 0.01; *p > 0.05.

Fig. 1 : LAMAP harbors conserved region domains of the AP endonuclease superfamily. (A) Multiple amino acid (aa) sequences alignment of members of the AP endonuclease superfamily from Escherichia coli and human (AP1) and corresponding sequences found in trypanosomatids after BLAST using the Leishmania amazonensis putative AP endonuclease (LAMAP) sequence as template. Identical amino acids are highlighted in black and similar in gray. The main residue block of aa presented in all organisms aligned (LQETK) is highlighted in red. Asterisks indicate residues responsible for nuclear localisation signals. (B) Phylogenetic tree constructed by neighbor-joining of exonucleases/endonucleases/phosphatases superfamily aa sequences. ENDO III: Endonuclease III. ENDO IV: Endonuclease IV. EXO III: Exonuclease III. Human AP1: human AP Endonuclease 1. Bootstrap values are displayed on each tree branch.

Fig. 2 : LAMAP restores DNA repair activity in Escherichia coli Base Excision Repair deficient strains. (A) E. coli wild type strain AB1157 (gray column) triple null mutant BW535 (white column) and triple mutant transformed with pLamap (black column) challenged with increasing concentrations of menadione. (B) E. coli triple null mutant BW535 (gray column) and triple mutant transformed with pLamap (black column) challenged with increasing concentrations of aminofluorene. p > 0.001; p > 0.01; p > 0.05.

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Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

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[1] + Corresponding author: bello@uerj.br