Structural basis for DNA recognition and nuclease processing by the Mre11 homologue SbcD in double-strand breaks repair

2014 ◽  
Vol 70 (2) ◽  
pp. 299-309 ◽  
Author(s):  
Shun Liu ◽  
Li-fei Tian ◽  
Yan-ping Liu ◽  
Xiao-min An ◽  
Qun Tang ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Catalytic Mechanism ◽  
Double Strand Breaks ◽  
Structural Basis ◽  
State Transitions ◽  
Endonuclease Activity ◽  
High Concentration ◽  
Binding Model ◽  
Strand Breaks ◽  
Mre11 Complex

The Mre11 complex comprising meiotic recombination 11 (Mre11), Rad50 and Nijmegen breakage syndrome 1 (Nbs1) plays multiple important roles in the sensing, processing and repair of DNA double-strand breaks (DSBs). Here, crystal structures of theEscherichia coliMre11 homologue SbcD and its Mn2+complex are reported. Dimerization of SbcD depends on a four-helix bundle consisting of helices α2, α3, α2′ and α3′ of the two monomers, and the irregular and bent conformation of helices α3 and α3′ in the SbcD dimer results in a dimeric arrangement that differs from those of previously reported Mre11 dimers. This finding indicates a distinct selectivity in DNA substrate recognition. The biochemical data combined with the crystal structures revealed that the SbcD monomer exhibits single-stranded DNA (ssDNA) endonuclease activity and double-stranded DNA (dsDNA) exonuclease activity on the addition of a high concentration of Mn2+. For the first time, atomic force microscopy analysis has been used to demonstrate that the SbcD monomer also possesses Mn2+-dependent dsDNA endonuclease activity. Loop β7–α6 of SbcD is likely to be a molecular switch and plays an important role in the regulation of substrate binding, catalytic reaction and state transitions. Based on structural and mutational analyses, a novel ssDNA-binding model of SbcD is proposed, providing insight into the catalytic mechanism of DSBs repair by the Mre11 complex.

scholarly journals Genetic Separation of Sae2 Nuclease Activity from Mre11 Nuclease Functions in Budding Yeast

2017 ◽  
Vol 37 (24) ◽  
Author(s):  
Sucheta Arora ◽  
Rajashree A. Deshpande ◽  
Martin Budd ◽  
Judy Campbell ◽  
America Revere ◽  
...  
Keyword(s):  
Nuclease Activity ◽  
Double Strand Breaks ◽  
Endonuclease Activity ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Content Type ◽  
Strand Breaks ◽  
Dna Ends

ABSTRACT Sae2 promotes the repair of DNA double-strand breaks in Saccharomyces cerevisiae. The role of Sae2 is linked to the Mre11/Rad50/Xrs2 (MRX) complex, which is important for the processing of DNA ends into single-stranded substrates for homologous recombination. Sae2 has intrinsic endonuclease activity, but the role of this activity has not been assessed independently from its functions in promoting Mre11 nuclease activity. Here we identify and characterize separation-of-function mutants that lack intrinsic nuclease activity or the ability to promote Mre11 endonucleolytic activity. We find that the ability of Sae2 to promote MRX nuclease functions is important for DNA damage survival, particularly in the absence of Dna2 nuclease activity. In contrast, Sae2 nuclease activity is essential for DNA repair when the Mre11 nuclease is compromised. Resection of DNA breaks is impaired when either Sae2 activity is blocked, suggesting roles for both Mre11 and Sae2 nuclease activities in promoting the processing of DNA ends in vivo. Finally, both activities of Sae2 are important for sporulation, indicating that the processing of meiotic breaks requires both Mre11 and Sae2 nuclease activities.

scholarly journals Structure based protein engineering to confer selectivity of guanine deaminase

2014 ◽  
Vol 70 (a1) ◽  
pp. C437-C437
Author(s):  
Aruna Bitra ◽  
Ruchi Anand
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Catalytic Efficiency ◽  
Structural Basis ◽  
Active Site Residues ◽  
X Ray ◽  
Secondary Activity ◽  
Guanine Deaminase ◽  
Differential Selectivity

Guanine deaminases (GDs) are important enzymes involved in both purine metabolism and nucleotide anabolism pathways. Here we present the molecular and catalytic mechanism of NE0047 and use the information obtained to engineer specific enzyme activities. NE0047 from Nitrosomonas europaea was found to be a high fidelity guanine deaminase (catalytic efficiency of 1.2 × 105 M–1 s–1). However; it exhibited secondary activity towards the structurally non-analogous triazine based compound ammeline. The X-ray structure of NE0047 in the presence of the substrate analogue 8-azaguanine help establish that the enzyme exists as a biological dimer and both the proper closure of the C-terminal loop and cross talk via the dimeric interface is crucial for conferring catalytic activity. It was further ascertained that the highly conserved active site residues Glu79 and Glu143 facilitate the deamination reaction by serving as proton shuttles. Moreover, to understand the structural basis of dual substrate specificity, X-ray structures of NE0047 in complex with a series of nucleobase analogs, nucleosides and substrate ammeline were determined. The crystal structures demonstrated that any substitutions in the parent substrates results in the rearrangement of the ligand in a catalytically unfavorable orientation and also impede the closure of catalytically important loop, thereby abrogating activity. However, ammeline was able to adopt a catalytically favorable orientation which, also allowed for proper loop closure. Based on the above knowledge of the crystal structures and the catalytic mechanism, the active site was subsequently engineered to fine-tune NE0047 activity. The mutated versions of the enzyme were designed so that they can function either exclusively as a GD or serve as specific ammeline deaminases. For example, mutations in the active site E143D and N66A confer the enzyme to be an unambiguous GD with no secondary activity towards ammeline. On the other hand, the N66Q mutant of NE0047 only deaminates ammeline. Additionally, a series of crystal structures of the mutant versions were solved that shed light on the structural basis of this differential selectivity.

scholarly journals A conserved Ctp1/CtIP C-terminal peptide stimulates Mre11 endonuclease activity

2021 ◽  
Vol 118 (11) ◽  
pp. e2016287118
Author(s):  
Aleksandar Zdravković ◽  
James M. Daley ◽  
Arijit Dutta ◽  
Tatsuya Niwa ◽  
Yasuto Murayama ◽  
...  
Keyword(s):  
Amino Acids ◽  
Homologous Recombination ◽  
Schizosaccharomyces Pombe ◽  
Double Strand Breaks ◽  
Pivotal Role ◽  
Endonuclease Activity ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
C Terminus ◽  
Dna Strands

The Mre11-Rad50-Nbs1 complex (MRN) is important for repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). The endonuclease activity of MRN is critical for resecting 5′-ended DNA strands at DSB ends, producing 3′-ended single-strand DNA, a prerequisite for HR. This endonuclease activity is stimulated by Ctp1, the Schizosaccharomyces pombe homolog of human CtIP. Here, with purified proteins, we show that Ctp1 phosphorylation stimulates MRN endonuclease activity by inducing the association of Ctp1 with Nbs1. The highly conserved extreme C terminus of Ctp1 is indispensable for MRN activation. Importantly, a polypeptide composed of the conserved 15 amino acids at the C terminus of Ctp1 (CT15) is sufficient to stimulate Mre11 endonuclease activity. Furthermore, the CT15 equivalent from CtIP can stimulate human MRE11 endonuclease activity, arguing for the generality of this stimulatory mechanism. Thus, we propose that Nbs1-mediated recruitment of CT15 plays a pivotal role in the activation of the Mre11 endonuclease by Ctp1/CtIP.

scholarly journals Coordinated nuclease activities counteract Ku at single-ended DNA double-strand breaks

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Pauline Chanut ◽  
Sébastien Britton ◽  
Julia Coates ◽  
Stephen P. Jackson ◽  
Patrick Calsou
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Double Strand Breaks ◽  
Exonuclease Activity ◽  
Endonuclease Activity ◽  
Dna Double Strand Breaks ◽  
Additional Mechanism ◽  
Strand Breaks ◽  
Rad51 Focus ◽  
Dna Resection

Abstract Repair of single-ended DNA double-strand breaks (seDSBs) by homologous recombination (HR) requires the generation of a 3′ single-strand DNA overhang by exonuclease activities in a process called DNA resection. However, it is anticipated that the highly abundant DNA end-binding protein Ku sequesters seDSBs and shields them from exonuclease activities. Despite pioneering works in yeast, it is unclear how mammalian cells counteract Ku at seDSBs to allow HR to proceed. Here we show that in human cells, ATM-dependent phosphorylation of CtIP and the epistatic and coordinated actions of MRE11 and CtIP nuclease activities are required to limit the stable loading of Ku on seDSBs. We also provide evidence for a hitherto unsuspected additional mechanism that contributes to prevent Ku accumulation at seDSBs, acting downstream of MRE11 endonuclease activity and in parallel with MRE11 exonuclease activity. Finally, we show that Ku persistence at seDSBs compromises Rad51 focus assembly but not DNA resection.

scholarly journals Structural basis for multifunctional roles of human Ints3 C-terminal domain

2020 ◽  
pp. jbc.RA120.016393
Author(s):  
Jian Li ◽  
Xinli Ma ◽  
Surajit Banerjee ◽  
Sankar Baruah ◽  
Nicholas J Schnicker ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Genome Stability ◽  
Double Strand Breaks ◽  
Structural Basis ◽  
Nucleic Acid Binding ◽  
Ataxia Telangiectasia Mutated ◽  
Interacting Protein ◽  
Strand Breaks ◽  
Terminal Domain

Proper repair of damaged DNA is critical for the maintenance of genome stability. A complex composed of Integrator subunit 3 (Ints3), single-stranded DNA-binding protein 1 (SSB1) and SSB-interacting protein 1 (SSBIP1) is required for efficient homologous recombination-dependent repair of double-strand breaks (DSBs) and ataxia-telangiectasia mutated (ATM)-dependent signaling pathways. It is known that in this complex the Ints3 N-terminal domain scaffolds SSB1 and SSBIP1. However, the molecular basis for the function of the Ints3 C-terminal domain remains unclear. Here, we present the crystal structure of the Ints3 C-terminal domain, uncovering a HEAT-repeat superhelical fold. Using structure and mutation analysis, we show that the C-terminal domain exists as a stable dimer. A basic groove and a cluster of conserved residues on two opposite sides of the dimer bind single-stranded RNA/DNA (ssRNA/ssDNA) and Integrator complex subunit 6 (Ints6), respectively. Dimerization is required for nucleic acid binding, but not for Ints6 binding. Additionally, in vitro experiments using HEK 293T cells demonstrate that Ints6 interaction is critical for maintaining SSB1 protein level. Taken together, our findings establish the structural basis of a multifunctional Ints3 C-terminal module, allowing us to propose a novel mode of nucleic acid recognition by helical repeat protein and paving the way for future mechanistic studies.

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