Abstract
The ATP binding cassette (ABC) proteins make up a large superfamily with members coming from all kingdoms. The functional form of the ABC protein nucleotide binding domain (NBD) is dimeric with ATP binding sites shared between subunits. The NBD is defined by six motifs: the Walker A, Q-loop, Signature, Walker-B, D-loop, and H-loop. The D-loop contains a conserved aspartate whose function is not clear but has been proposed to be involved in cross-talk between ATP binding sites. Structures of various ABC proteins suggest an interaction between the D-loop aspartate and an asparagine residue located in Walker A loop of the opposing subunit. Here, we evaluate the functional role of the D-loop using a bacteriophage T4 ABC protein, Rad50 (gp46). Mutation of either the D-loop aspartate or the Walker A asparagine results in dramatic reductions in ATP affinity, hydrolysis rate, and cooperativity. The mutant proteins bind Mre11 (gp47) and DNA normally, but no longer support the ATP-dependent nuclease activities of Mre11. We propose that the D-loop aspartate functions to stabilize the Walker A asparagine in a position favorable for catalysis. We find that the asparagine is crucially important to the mechanism of ATP hydrolysis by increasing the affinity for ATP and positioning the γ-phosphate of ATP for catalysis. Additionally, we propose that the asparagine acts as a γ-phosphate sensor and, through its interaction with the conserved D-loop aspartate, transmits conformational changes across the dimer interface to the second ATP binding site.
Highlights
Rad50 and Mre11 form a heterotetrameric complex (Mre112/ Rad502, referred to as the MR2 complex) that is involved in DNA double-strand break (DSB) repair [1]
The MR complex is involved in the first step of homologous recombination (HR), which is the generation of a 3Ј singlestranded DNA in a process referred to as DSB resection
Bacteria contain an MR homolog, it appears that it is not involved in DSB resection but instead functions to prevent of cruciform structures that have the potential to block the replication fork [15]
Summary
Materials—Oligodeoxynucleotides used for mutagenesis were from the Iowa State University DNA Facility. The Rad protein was eluted in buffer containing 20 mM Tris-Cl, 200 mM NaCl, 150 mM imidazole, 10% glycerol (v/v), pH 8.0 (4 °C). The column was washed with 100 ml of equilibration buffer and the Rad protein was eluted with 20 mM Tris-Cl, 400 mM NaCl, 20% glycerol (v/v), pH 8.0 (4 °C). Mre Pull-down Assay—Purified Mre (10 g) and purified WT or mutant Rad proteins (10 g) were incubated for 10 min at 22 °C in 100 l of pull-down buffer (20 mM Tris-Cl, 500 mM NaCl, 5 mM imidazole, 10% glycerol (v/v), pH 8.0). For the 17th position DNA substrate, rates were determined over the initial 5 min of the reaction for the MR complex (400 nM complex) in both the absence and presence of ATP
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