Modes of action of the archaeal Mre11/Rad50 DNA-repair complex revealed by fast-scan atomic force microscopy

Zabolotnaya, Ekaterina; Mela, Ioanna; Williamson, Mark J.; Bray, Sian; Yau, Siu Kei; Papatziamou, Dimitra; Edwardson, J. Michael; Robinson, Nicholas P.; Henderson, Ronald W.

doi:10.1073/pnas.1915598117

Cited by 17 publications

(28 citation statements)

References 70 publications

(125 reference statements)

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“…This dynamic DNA end-surveillance system, dependent on chromosomal-tracking revolutionized our views of DSB recognition by the M/R complex. Furthermore, in support of this model, a recent study of the M/R complex from the thermophilic archaeon Sulfolobus acidocaldarius , using real-time AFM, has demonstrated that the zinc-hook region of Rad50 appears to associate transiently with DNA substrates [ 43 ]. This study also predicted, by classical molecular dynamics simulations of the archaeal Rad50 hinged region on double-stranded DNA, that the zinc-hook region and juxtaposed coiled coils are seemingly able to track the minor groove of a DNA duplex until an end is encountered [ 43 ].…”

Section: Comparisons Of the Analogous Rad50 Zinc Hook And Smc Hinge Rmentioning

confidence: 95%

“…These duplex strand-separations might conceivably arise as a consequence of supercoiling or writhe generated from the energy-dependent manipulation of the DNA substrate by the archaeal M/R complex [43]. Fast-scan AFM analyzes in fluid also revealed that archaeal M/R complex can manipulate DNA substrates in a manner consistent with translocation and loop formation [43]. While further experimental interrogation is required to verify how the M/R complexes are involved in the active manipulation of DNA substrates, the mounting data provided by studies of the canonical SMC proteins indicate that the ATP-dependent architectural contortions induced by these complexes are indeed linked to DNA translocation and chromosomal loop extrusion [43,68,69,[74][75][76][77]80].…”

Section: Comparisons Of the Analogous Rad50 Zinc Hook And Smc Hinge Rmentioning

confidence: 99%

“…The Mre11 nuclease (blue) interacts with the Rad50 catalytic domains via the ‘capping’ domain (Cap) and helix–loop–helix regions (hlh), which extend from the core nucleolytic domain (Nuc). ( B – E ) The M/R complex typically arranges as a tetramer (although higher order assemblies are possible [ 42 , 43 ], either via interactions at the zinc-hook ( B ) or by association of the catalytic globular domains ( C , D ). The ring-shaped arrangement can form when the intramolecular associations are simultaneously formed at both the Rad50 zinc-hooks and the Mre11 globular domains ( D ).…”

Section: Boiling Down the Mechanisms Of M/r Action: Insight Gleaned Fmentioning

confidence: 99%

“…The zinc-binding element, distal to the catalytic domains, is commonly referred to as the 'zinc-hook' and is analogous to the centrally located 'hinge' region of other SMC proteins [38][39][40]. M/R tetramers can remain associated via these zinc hook regions when the globular domains have separated ( Figure 2B) [41][42][43]. It has also been suggested, based on atomic force microscopy (AFM) and electron microscopy studies that these zinc-hook regions play critical roles in mediating chromosomal bridging via intermolecular associations, with the hooks associating between reciprocal Rad50 molecules [33,38,41,42,44,45].…”

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confidence: 99%

“…The Mre11 nuclease (blue) interacts with the Rad50 catalytic domains via the 'capping' domain (Cap) and helix-loop-helix regions (hlh), which extend from the core nucleolytic domain (Nuc). (B-E) The M/R complex typically arranges as a tetramer (although higher order assemblies are possible [42,43], either via interactions at the inflexible linear rods. Such changes permit the zinc hooks at the apices to associate intermolecularly with other M/R complexes in a manner that has been proposed to support DNA bridging during the repair events [41] ( Figure 2E).…”

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confidence: 99%

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Turning the Mre11/Rad50 DNA repair complex on its head: lessons from SMC protein hinges, dynamic coiled-coil movements and DNA loop-extrusion?

Zabolotnaya

Mela

Henderson

et al. 2020

Biochemical Society Transactions

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The bacterial SbcC/SbcD DNA repair proteins were identified over a quarter of a century ago. Following the subsequent identification of the homologous Mre11/Rad50 complex in the eukaryotes and archaea, it has become clear that this conserved chromosomal processing machinery is central to DNA repair pathways and the maintenance of genomic stability in all forms of life. A number of experimental studies have explored this intriguing genome surveillance machinery, yielding significant insights and providing conceptual advances towards our understanding of how this complex operates to mediate DNA repair. However, the inherent complexity and dynamic nature of this chromosome-manipulating machinery continue to obfuscate experimental interrogations, and details regarding the precise mechanisms that underpin the critical repair events remain unanswered. This review will summarize our current understanding of the dramatic structural changes that occur in Mre11/Rad50 complex to mediate chromosomal tethering and accomplish the associated DNA processing events. In addition, undetermined mechanistic aspects of the DNA enzymatic pathways driven by this vital yet enigmatic chromosomal surveillance and repair apparatus will be discussed. In particular, novel and putative models of DNA damage recognition will be considered and comparisons will be made between the modes of action of the Rad50 protein and other related ATPases of the overarching SMC superfamily.

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Section: Comparisons Of the Analogous Rad50 Zinc Hook And Smc Hinge Rmentioning

confidence: 95%

Section: Comparisons Of the Analogous Rad50 Zinc Hook And Smc Hinge Rmentioning

confidence: 99%

Section: Boiling Down the Mechanisms Of M/r Action: Insight Gleaned Fmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Turning the Mre11/Rad50 DNA repair complex on its head: lessons from SMC protein hinges, dynamic coiled-coil movements and DNA loop-extrusion?

Zabolotnaya

Mela

Henderson

et al. 2020

Biochemical Society Transactions

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Three segment ligation of a 104 kDa multi-domain protein by SrtA and OaAEP1

2022

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NMR spectroscopy is an excellent tool for studying protein structure and dynamics which provides a deeper understanding of biological function. As the size of the biomolecule of interest increases, it can become advantageous to dilute the number of observed signals in the NMR spectrum to decrease spectral overlap and increase resolution. One way to limit the number of resonances in the NMR data is by selectively labeling a smaller domain within the larger macromolecule, a process called segmental isotopic labeling. Many examples of segmental isotopic labeling have been described where two segments of a protein are ligated together by chemical or enzymatic means, but there are far fewer descriptions of a three or more segment ligation reaction. Herein, we describe an enzymatic segmental labeling scheme that combines the widely used Sortase A and more recently described OaAEP1 for a two site ligation strategy. In preparation to study proposed long-range allostery in the 104 kDa DNA damage repair protein Rad50, we ligated side-chain methyl group labeled Zn Hook domain between two long segments of otherwise unlabeled P.furiosus Rad50. Enzymatic activity data demonstrated that the scars resulting from the ligation reactions did not affect Rad50 function within the Mre11-Rad50 DNA double strand break repair complex. Finally, methyl-based NMR spectroscopy confirmed the formation of the full-length ligated protein. Our strategy highlights the strengths of OaAEP1 for segmental labeling, namely faster reaction times and a smaller recognition sequence, and provides a straightforward template for using these two enzymes in multisite segmental labeling reactions.

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