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Functional characterization of the putative Aspergillus nidulans DNA damage binding protein homologue DdbA

Abstract

Nucleotide excision repair (NER) eliminates helix-distorting DNA base lesions. Seven XP-deficient genetic complementation groups (XPA to XPG) have already been identified in mammals, and their corresponding genes have been cloned. Hereditary defects in NER are associated with several diseases, including xeroderma pigmentosum (XP). UV-DDB (XPE) is formed by two associated subunits, DDB1 and DDB2. UV-DDB was identified biochemically as a protein factor that exhibits very strong and specific binding to ultraviolet (UV)-treated DNA. As a preliminary step to characterize the components of the NER in the filamentous fungus Aspergillus nidulans, here we identified a putative DDB1 homologue, DdbA. Deletion and expression analysis indicated that A. nidulans ddbA gene is involved in the DNA damage response, more specifically in the UV light response and 4-nitroquinoline oxide (4-NQO) sensitivity. Furthermore, the ΔddbA strain cannot self-cross and expression analysis showed that ddbA can be induced by oxidative stress and is developmentally regulated in both asexual and sexual processes. The ΔddbA mutation can genetically interact with uvsB ATR, atmAATM, nkuA KU70, H2AX-S129A (a replacement of the conserved serine in the C-terminal of H2AX with alanine), and cshB (a mutation in CSB Cockayne’s syndrome protein involved in the transcription-coupled repair subpathway of NER) mutations. Finally, to determine the DdbA cellular localization, we constructed a GFP::DdbA strain. In the presence and absence of DNA damage, DdbA was mostly detected in the nuclei, indicating that DdbA localizes to nuclei and its cellular localization is not affected by the cellular response to DNA damage induced by 4-NQO and UV light.

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Acknowledgments

This research was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil, and John Guggenheim Memorial Foundation (USA). We also thank Drs. Steve A. Osmani and Collin P. de Souza for providing the H2AX-S129A mutant strain, the two anonymous reviewers and Dr. Andres Aguilera for their comments, and the Laboratory of Confocal Microscopy, Faculty of Medicine, University of São Paulo, Brazil, for access to the confocal microscope.

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Correspondence to Gustavo Henrique Goldman.

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Communicated by A. Aguilera.

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Lima, J.F., Malavazi, I., da Silva Ferreira, M.E. et al. Functional characterization of the putative Aspergillus nidulans DNA damage binding protein homologue DdbA. Mol Genet Genomics 279, 239–253 (2008). https://doi.org/10.1007/s00438-007-0307-0

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Keywords

  • Aspergillus nidulans
  • Nucleotide Excision Repair
  • DDB1
  • DNA damage binding protein