Functional characterization of the putative Aspergillus nidulans DNA damage binding protein homologue DdbA.
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 DeltaddbA 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 DeltaddbA mutation can genetically interact with uvsB (ATR), atmA(ATM), 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.
Lima
Joel Fernandes
JF
Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café S/N, CEP 14040-903, Ribeirão Preto, São Paulo, Brazil.
Malavazi
Iran
I
da Silva Ferreira
Márcia Eliana
ME
Savoldi
Marcela
M
Mota
André Oliveira
AO
Capellaro
José Luiz
JL
de Souza Goldman
Maria Helena
MH
Goldman
Gustavo Henrique
GH
D015112:4-Nitroquinoline-1-oxide / Q000494:pharmacology; D001233:Aspergillus nidulans / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism* / Q000528:radiation effects; D001483:Base Sequence; D004249:DNA Damage; D004260:DNA Repair; D004271:DNA, Fungal / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D025141:Drug Resistance, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D005800:Genes, Fungal; D009154:Mutation; D018384:Oxidative Stress; D010802:Phylogeny; D011836:Radiation Tolerance / Q000235:genetics; D014466:Ultraviolet Rays
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D015112:4-Nitroquinoline-1-oxide