Abstract
The Mre11-Rad50 (MR) complex is a central player in DNA repair and is implicated in the processing of DNA ends caused by double strand breaks. Recent crystal structures of the MR complex suggest that several conformational rearrangements occur during its ATP hydrolysis cycle. A comparison of the Mre11 dimer interface from these structures suggests that the interface is dynamic in nature and may adopt several different arrangements. To probe the functional significance of the Mre11 dimer interface, we have generated and characterized a dimer disruption Mre11 mutant (L101D-Mre11). Although L101D-Mre11 binds to Rad50 and dsDNA with affinity comparable with the wild-type enzyme, it does not activate the ATP hydrolysis activity of Rad50, suggesting that the allosteric communication between Mre11 and Rad50 has been interrupted. Additionally, the dsDNA exonuclease activity of the L101D-MR complex has been reduced by 10-fold under conditions where processive exonuclease activity is required. However, we unexpectedly found that under steady state conditions, the nuclease activity of the L101D-MR complex is significantly greater than that of the wild-type complex. Based on steady state and single-turnover nuclease assays, we have assigned the rate-determining step of the steady state nuclease reaction to be the productive assembly of the complex at the dsDNA end. Together, our data suggest that the Mre11 dimer interface adopts at least two different states during the exonuclease reaction.
Highlights
The Mre11 dimer interface may be dynamic, responding to ATP and/or DNA
In an effort to determine the significance of the dimer interface, we investigated the effects of the mutation on the MR complex in terms of Rad50 and DNA affinity, activation of Rad50 ATP hydrolysis activity, and nuclease activity
X-ray crystal structures and protein-protein cross-linking experiments have demonstrated that ATP binding and/or hydrolysis by Rad50 affects the conformation of Mre11 [25, 27]
Summary
The Mre dimer interface may be dynamic, responding to ATP and/or DNA. Results: A mutation in the Mre dimer interface increases the nuclease initiation rate but decreases the translocation rate and lowers processivity. This interface is defined by a shared Zn2ϩ-binding site that is formed through a tetrathiolate linkage where each coiled coil provides two cysteine residues This type of binding site generally binds Zn2ϩ with extremely high affinity and is thought to be extremely stable, atomic force microscopy experiments suggest that the Rad zinc-hook domain is dynamic and able to respond to DNA binding by Rad50 [30]. X-ray crystal structures and protein cross-linking indicate that the Mre dimeric interface undergoes small conformation rearrangements upon ATP binding by Rad50 [24, 27]. In an effort to determine the significance of the dimer interface, we investigated the effects of the mutation on the MR complex in terms of Rad and DNA affinity, activation of Rad ATP hydrolysis activity, and nuclease activity. The disruption has profound effects on the ATPase activity of Rad and the exonuclease activity of Mre in both steady state and pre-steady state assays
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