Faculty Opinions recommendation of Structures and single-molecule analysis of bacterial motor nuclease AdnAB illuminate the mechanism of DNA double-strand break resection.

2019 ◽  
Author(s):  
Charles Bell
Keyword(s):  
Single Molecule ◽  
Strand Break ◽  
Double Strand Break ◽  
Dna Double Strand Break ◽  
Single Molecule Analysis

scholarly journals Structures and single-molecule analysis of bacterial motor nuclease AdnAB illuminate the mechanism of DNA double-strand break resection

2019 ◽  
Vol 116 (49) ◽  
pp. 24507-24516 ◽  
Author(s):  
Ning Jia ◽  
Mihaela C. Unciuleac ◽  
Chaoyou Xue ◽  
Eric C. Greene ◽  
Dinshaw J. Patel ◽  
...  
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Iron Sulfur Cluster ◽  
Strand Breaks ◽  
Nuclease Domain ◽  
Dna Double Strand Break ◽  
Before And After ◽  
Single Molecule Analysis

Mycobacterial AdnAB is a heterodimeric helicase–nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Here we report cryoelectron microscopy (cryo-EM) structures of AdnAB in three functional states: in the absence of DNA and in complex with forked duplex DNAs before and after cleavage of the 5′ single-strand DNA (ssDNA) tail by the AdnA nuclease. The structures reveal the path of the 5′ ssDNA through the AdnA nuclease domain and the mechanism of 5′ strand cleavage; the path of the 3′ tracking strand through the AdnB motor and the DNA contacts that couple ATP hydrolysis to mechanical work; the position of the AdnA iron–sulfur cluster subdomain at the Y junction and its likely role in maintaining the split trajectories of the unwound 5′ and 3′ strands. Single-molecule DNA curtain analysis of DSB resection reveals that AdnAB is highly processive but prone to spontaneous pausing at random sites on duplex DNA. A striking property of AdnAB is that the velocity of DSB resection slows after the enzyme experiences a spontaneous pause. Our results highlight shared as well as distinctive properties of AdnAB vis-à-vis the RecBCD and AddAB clades of bacterial DSB-resecting motor nucleases.

scholarly journals Dissection of DNA double-strand-break repair using novel single-molecule forceps

2018 ◽  
Vol 25 (6) ◽  
pp. 482-487 ◽  
Author(s):  
Jing L. Wang ◽  
Camille Duboc ◽  
Qian Wu ◽  
Takashi Ochi ◽  
Shikang Liang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Dna Double Strand Break ◽  
Break Repair

scholarly journals Single-molecule analysis reveals cooperative stimulation of Rad51 filament nucleation and growth by mediator proteins

2020 ◽  
Author(s):  
Ondrej Belan ◽  
Consuelo Barroso ◽  
Artur Kaczmarczyk ◽  
Roopesh Anand ◽  
Stefania Federico ◽  
...  
Keyword(s):  
Dna Damage ◽  
Single Molecule ◽  
Strand Break ◽  
Nucleation And Growth ◽  
Repair Mechanism ◽  
Dna Double Strand Break ◽  
Strand Invasion ◽  
Single Molecule Analysis ◽  
Stimulation Of

ABSTRACTHomologous recombination (HR) is an essential DNA double-strand break (DSBs) repair mechanism frequently inactivated in cancer. During HR, RAD51 forms nucleoprotein filaments on RPA-coated resected DNA and catalyses strand invasion into homologous duplex DNA. How RAD51 displaces RPA and assembles into long HR-proficient filaments remains uncertain. Here, we employ single-molecule imaging to investigate the mechanism of nematode RAD-51 filament growth in the presence of BRC-2 (BRCA2) and RAD-51 paralogs, RFS-1/RIP-1. BRC-2 nucleates RAD-51 on RPA-coated DNA, while RFS-1/RIP-1 acts as a ‘chaperone’ to promote 3’ to 5’ filament growth via highly dynamic engagement with 5’ filament ends. Inhibiting ATPase or mutation in RFS-1 Walker box leads to RFS-1/RIP-1 retention on RAD-51 filaments and hinders growth. rfs-1 Walker box mutants display sensitivity to DNA damage and accumulate RAD-51 complexes non-functional for HR in vivo. Our work reveals the mechanism of RAD-51 nucleation and filament growth in the presence of recombination mediators.

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