RecA-like domain 2 of DNA-dependent ATPase A domain, a SWI2/SNF2 protein, mediates conformational integrity and ATP hydrolysis

Abstract

ATP-dependent chromatin remodeling proteins use the energy released from ATP hydrolysis to reposition nucleosomes in DNA-dependent processes. These proteins are classified as SF2 helicases. SMARCAL1, a member of this protein family, is known to modulate both DNA repair and transcription by specifically recognizing DNA molecules possessing double-strand to single-strand transition regions. Mutations in this gene cause a rare autosomal recessive disorder known as Schimke Immuno-Osseous Dysplasia (SIOD).Structural studies have shown that the ATP-dependent chromatin remodeling proteins possess two RecA-like domains termed as RecA-like domain 1 and RecA-like domain 2. Using Active DNA-dependent ATPase A domain (ADAAD), the bovine homolog of SMARCAL1, as a model system we had previously shown that the RecA-like domain 1 containing helicase motifs Q, I, Ia, II, and III are sufficient for ligand binding; however, the Rec A-like domain 2 containing motifs IV, V, and VI are needed for ATP hydrolysis. In the present study, we have focused on the motifs present in the RecA-like domain 2. Our studies demonstrate that the presence of an aromatic residue in motif IV is needed for interaction with DNA in the presence of ATP. We also show that the motif V is required for the catalytic efficiency of the protein and motif VI is needed for interaction with DNA in the presence of ATP. Finally, we show that the SIOD-associated mutation, R820H, present in motif VI results in loss of ATPase activity, and therefore, reduced response to DNA damage.

SMARCAL1, the annealing helicase and the transcriptional co‐regulator

“…40 In ADAAD, the function of this motif has been studied in combination with that of motif VI and is discussed later in that section. 41 The motif III contains S/GTP sequence and has been shown to couple ATP hydrolysis to strand separation in PcrA, an SF1 family helicase. 42 In Ded1 helicase belonging to the SF2 family this motif has been shown to be required for RNA-dependent ATPase activity but not for high affinity ATP binding.…”

“…Mutations in these motifs alter the conformation of the protein; however, the stability of the protein is not affected. 41 In SMARCAL1, the motif IV contains FAHH sequence where the aromatic amino acid is important for both ATP and DNA binding. 41 Mutation of F to A in ADAAD disrupts both DNA as well as ATP binding.…”

“…41 In SMARCAL1, the motif IV contains FAHH sequence where the aromatic amino acid is important for both ATP and DNA binding. 41 Mutation of F to A in ADAAD disrupts both DNA as well as ATP binding. Replacing the F with another aromatic amino acid restores ATP hydrolysis with altered kinetics, 41 thus suggesting that the presence of an aromatic residue is necessary at this position.…”

“…41 Mutation of F to A in ADAAD disrupts both DNA as well as ATP binding. Replacing the F with another aromatic amino acid restores ATP hydrolysis with altered kinetics, 41 thus suggesting that the presence of an aromatic residue is necessary at this position. In Ded1 helicase too, the F residue has been shown to be important at this position.…”

“…40 However, in SMARCAL1, this sequence is defined as SITANN where mutating either the S or the T residue results in ATPase active mutant that shows altered kinetics. 41 The motif, thus, appears to be dictating the catalytic efficiency of the protein.…”