Abstract
The Mre11-Rad50 complex (MR) from bacteriophage T4 (gp46/47) is involved in the processing of DNA double-strand breaks. Here, we describe the activities of the T4 MR complex and its modulation by proteins involved in homologous recombination. T4 Mre11 is a Rad50- and Mn(2+)-dependent dsDNA exonuclease and ssDNA endonuclease. ATP hydrolysis is required for the removal of multiple nucleotides via dsDNA exonuclease activity but not for the removal of the first nucleotide or for ssDNA endonuclease activity, indicating ATP hydrolysis is only required for repetitive nucleotide removal. By itself, Rad50 is a relatively inefficient ATPase, but the presence of Mre11 and dsDNA increases ATP hydrolysis by 20-fold. The ATP hydrolysis reaction exhibits positive cooperativity with Hill coefficients ranging from 1.4 for Rad50 alone to 2.4 for the Rad50-Mre11-DNA complex. Kinetic assays suggest that approximately four nucleotides are removed per ATP hydrolyzed. Directionality assays indicate that the prevailing activity is a 3' to 5' dsDNA exonuclease, which is incompatible with the proposed role of MR in the production of 3' ssDNA ends. Interestingly, we found that in the presence of a recombination mediator protein (UvsY) and ssDNA-binding protein (gp32), Mre11 is capable of using Mg(2+) as a cofactor for its nuclease activity. Additionally, the Mg(2+)-dependent nuclease activity, activated by UvsY and gp32, results in the formation of endonuclease reaction products. These results suggest that gp32 and UvsY may alter divalent cation preference and facilitate the formation of a 3' ssDNA overhang, which is a necessary intermediate for recombination-mediated double-strand break repair.
Highlights
An mRNA transcript, which is processed and remains stably bound at the origin in what is known as an R-loop [2]
double-strand break (DSB) are repaired through two pathways, non-homologous end joining (NHEJ) and homologous recombination (HR); in T4 phage it appears that all DSBs are repaired through HR [4, 11]
The observed 3Ј to 5Ј dsDNA exonuclease activity is incompatible with its proposed in vivo function, we find that the presence of two T4 recombination proteins, UvsY and gp32, alter the nuclease activity of the MR complex, which may promote the formation of a 3Ј ssDNA recombinogenic end
Summary
Expression, and Purification of T4 Mre and wtRad and K42M-Rad50—The open reading frames for gp and 47 were PCR-amplified from bacteriophage T4 genomic DNA (Sigma) and cloned into the pTYB1 expression vector (New England Biolabs) using the NdeI and SapI restriction sites using the following primers: T4Rad50-NdeF, 5Ј-GGTGGTCATATGAAGAATTTTAAACTTAATAG; T4Rad50SapR, 5Ј-GGTGGTTGCTCTTCCGCATTAAACCATTACAGTAAATCG; T4Mre11-NdeF, 5Ј-GGTGGTCATATGAAAATTTTAAATTTAGGTG; T4Mre11-SapR, 5Ј-GGTGGTTGCTCTTCCGCATCATTGTGTTGCCTCTACATATAG. T4 Rad was eluted from the P11 column with P11 elution buffer (20 mM Tris-HCl, 400 mM NaCl, 20% glycerol, pH 8.0), concentrated to 25–50 M, dispensed into aliquots, and frozen at Ϫ80 °C. The reaction buffer contained 50 mM Tris-HCl, pH 7.6, 50 mM KCl, 5 mM MgCl2, 0.3 mM MnCl2, 0.1 M MR complex, and 1.3 M 2-AP labeled dsDNA. To determine the steady-state ATP hydrolysis activity of Rad and the MR complex, we employed the standard coupled fluorometric ATP hydrolysis assay [46] carried out at 30 °C using excitation and emission wavelengths of 340 and 460 nm, respectively, on either a SLM-Aminco (SLM Instruments, Inc.) or a Cary Eclipse spectrofluorometer (Varian). In reactions containing DNA, its concentration was held in excess over that of Rad or the MR complex and well above the Kactivation for DNA. Steady-state rate constants were determined using Sigmaplot 10.0/Enzyme Kinetics Module 1.3 (Systat Software, Inc.)
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