Ligand‐induced conformation changes drive ATP hydrolysis and function in SMARCAL1

Abstract

Mutations and deletions in SMARCAL1, an SWI2/SNF2 protein, cause Schimke immuno-osseous dysplasia (SIOD). SMARCAL1 preferentially binds to DNA molecules possessing double-stranded to single-stranded transition regions and mediates annealing helicase activity. The protein is critical for alleviating replication stress and maintaining genome integrity. In this study, we have analysed the ATPase activity of three mutations -A468P, I548N and S579L -present in SIOD patients. These mutations are present in RecA-like domain I of the protein. Analysis using active DNA-dependent ATPase A domain (ADAAD), an N-terminal deleted construct of bovine SMARCAL1, showed that all three mutants were unable to hydrolyse ATP. Conformational studies indicated that the a-helix and b-sheet content of the mutant proteins was altered compared to the wild-type protein. Molecular simulation studies confirmed that major structural changes had occurred in the mutant proteins. These changes included alteration of a loop region connecting motif Ia and II. As motif Ia has been implicated in DNA binding, ligand binding studies were done using fluorescence spectroscopy. These studies revealed that the K d for protein-DNA interaction in the presence of ATP was indeed altered in the case of mutant proteins compared to the wild-type. Finally, in vivo studies were done to complement the in vitro and in silico studies. The results from these experiments demonstrate that mutations in human SMARCAL1 that result in loss in ATPase activity lead to increased replication stress and therefore possibly manifestation of SIOD.

SMARCAL1, the annealing helicase and the transcriptional co‐regulator

“…43 The S residue of the motif III in ADAAD is mutated in SIOD patients and has been studied in this context as discussed later in the review. 44 The motifs IV, V, and VI lie in the Rec A-like domain 2 and are important for the conformation of the protein.…”

“…Mutation A468P that occurs on a residue present just one amino acid after motif I, mutation I548N that occurs on the residue present just before DESH box, and mutation S579L that occurs on the serine of motif III (SGTP) have been studied with respect to ATP hydrolysis and ligand binding. 44 All these three mutations result in impaired DNA binding in the presence of ATP such that the conformational change required for ATP hydrolysis does not occur leading to abrogated ATPase activity. 44 Motif VI associated mutation R870H results in altered conformation of the protein such that the mutant can no longer bind DNA either in the absence or presence of ATP with the same affinity as the wild-type protein.…”

“…44 All these three mutations result in impaired DNA binding in the presence of ATP such that the conformational change required for ATP hydrolysis does not occur leading to abrogated ATPase activity. 44 Motif VI associated mutation R870H results in altered conformation of the protein such that the mutant can no longer bind DNA either in the absence or presence of ATP with the same affinity as the wild-type protein. 41 All four mutations, thus, lead to loss in ATPase activity.…”

Regulation of ATM and ATR by SMARCAL1 and BRG1

“…We rationalized that if SMARCAL1 and BRG1 are necessary for DNA damage response pathway, then the patient-associated mutations should abrogate the DNA damage response. Therefore, the role of three SIOD-associated mutants-A468P, I548N, and S579L-was studied as previously it has been shown that these three mutants are unable to hydrolyze ATP due to impaired DNA binding [53]. In addition, three CSSassociated mutants-T895M, L921F, and M1011T-were also chosen as these three mutants mapped to the ATPase domain and are postulated to lack ATPase activity [27].…”

Annealing of Complementary DNA Sequences During Double-Strand Break Repair inDrosophilaIs Mediated by the Ortholog of SMARCAL1

“…It is possible that the Marcal1 K275M mutant protein may bind DNA less tightly than the wild-type protein. While ATP binding is not required for DNA binding by SMARCAL1, it can cause a conformational change that influences the DNA-binding constant (Gupta et al 2015). It is also possible that multiple Marcal1 molecules bind the nascent DNA and the presence of any wild-type protein is sufficient to rescue the Marcal1 K275M phenotype.…”