Genetic Interactions of the Aspergillus nidulans atmAATM Homolog With Different Components of the DNA Damage Response Pathway

Abstract

Ataxia telangiectasia mutated (ATM) is a phosphatidyl-3-kinase-related protein kinase that functions as a central regulator of the DNA damage response in eukaryotic cells. In humans, mutations in ATM cause the devastating neurodegenerative disease ataxia telangiectasia. Previously, we characterized the homolog of ATM (AtmA) in the filamentous fungus Aspergillus nidulans. In addition to its expected role in the DNA damage response, we found that AtmA is also required for polarized hyphal growth. Here, we extended these studies by investigating which components of the DNA damage response pathway are interacting with AtmA. The AtmA ATM loss of function caused synthetic lethality when combined with mutation in UvsB ATR . Our results suggest that AtmA and UvsB are interacting and they are probably partially redundant in terms of DNA damage sensing and/or repairing and polar growth. We identified and inactivated A. nidulans chkA CHK1 and chkB CHK2 genes. These genes are also redundantly involved in A. nidulans DNA damage response. We constructed several combinations of double mutants for DatmA, DuvsB, DchkA, and DchkB. We observed a complex genetic relationship with these mutations during the DNA replication checkpoint and DNA damage response. Finally, we observed epistatic and synergistic interactions between AtmA, and bimE APC1, ankA WEE1 and the cdc2-related kinase npkA, at S-phase checkpoint and in response to DNA-damaging agents.

Functional Analysis of the Aspergillus nidulans Kinome

“…As previously reported, uvsB ATR mutants were sensitive to a wide variety of genotoxic agents including the DNA alkylating agent DEO, the DNA double strand break inducing agent camptothecin and the DNA replication inhibitor HU [56], [57]. Contrasting this, the atmA A ™ mutant was only strongly sensitive to camptothecin, consistent with ATM kinase functions being more specific for double strand DNA breaks [31], [58], [59] (Figure 7). Both the chkA chk1 and chkB chk2 effector kinase mutants were sensitive to HU but not the DNA damaging agents tested (Figure 7).…”

“…Both the chkA chk1 and chkB chk2 effector kinase mutants were sensitive to HU but not the DNA damaging agents tested (Figure 7). As HU sensitivity was previously reported for chkA chk1 but not chkB chk2 mutants [59], we tested the viability of these mutants at different concentrations of HU (Figure S7 in File S1), confirming their HU sensitivity.…”

ATM and ATR homologes of Neurospora crassa are essential for normal cell growth and maintenance of chromosome integrity

“…While both uvsB and atmA disruptants are viable, the double mutant is inviable. Furthermore, conditional loss of atmA expression in a uvsB null mutant results in further increased mutagen sensitivity (Malavazi et al, 2008). These findings indicate that the relationship between uvsB and atmA is similar to the relationship between ATR and ATM in mammals.…”

“…The same relationship was observed in A. nidulans: the double mutant of atmA and uvsB cannot be obtained by crossing or gene replacement (Malavazi et al, 2008). In S. cerevisiae and mouse, ATR homologes are essential for cell growth or development, but ATM homologes are not (Morrow et al, 1995;Barlow et al, 1996;Brown and Baltimore, 2000).…”

“…Cells containing micronuclei (%) mus-21 mus-9 WT C mus-21 mus-9 ts mus-21 mus-9 WT mus-9 ts and Cortez, 2008;Malavazi et al, 2008;Garner and Costanzo, 2009). Thus, the relationship between two PIKKs may be conserved from filamentous fungi to vertebrates.…”