scholarly journals Meiosis-specific recombinase Dmc1 is a potent inhibitor of the Srs2 antirecombinase

2018 ◽  
Vol 115 (43) ◽  
pp. E10041-E10048 ◽  
Author(s):  
J. Brooks Crickard ◽  
Kyle Kaniecki ◽  
Youngho Kwon ◽  
Patrick Sung ◽  
Eric C. Greene
Keyword(s):  
Chromosome Segregation ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Potent Inhibitor ◽  
Chromosomal Rearrangements ◽  
Motor Protein ◽  
Product Formation ◽  
Gross Chromosomal Rearrangements ◽  
Strand Annealing ◽  
Hydrolysis Activity

Cross-over recombination products are a hallmark of meiosis because they are necessary for accurate chromosome segregation and they also allow for increased genetic diversity during sexual reproduction. However, cross-overs can also cause gross chromosomal rearrangements and are therefore normally down-regulated during mitotic growth. The mechanisms that enhance cross-over product formation upon entry into meiosis remain poorly understood. In Saccharomyces cerevisiae, the Superfamily 1 (Sf1) helicase Srs2, which is an ATP hydrolysis-dependent motor protein that actively dismantles recombination intermediates, promotes synthesis-dependent strand annealing, the result of which is a reduction in cross-over recombination products. Here, we show that the meiosis-specific recombinase Dmc1 is a potent inhibitor of Srs2. Biochemical and single-molecule assays demonstrate that Dmc1 acts by inhibiting Srs2 ATP hydrolysis activity, which prevents the motor protein from undergoing ATP hydrolysis-dependent translocation on Dmc1-bound recombination intermediates. We propose a model in which Dmc1 helps contribute to cross-over formation during meiosis by antagonizing the antirecombinase activity of Srs2.

scholarly journals Genetic Steps of Mammalian Homologous Repair with Distinct Mutagenic Consequences

2004 ◽  
Vol 24 (21) ◽  
pp. 9305-9316 ◽  
Author(s):  
Jeremy M. Stark ◽  
Andrew J. Pierce ◽  
Jin Oh ◽  
Albert Pastink ◽  
Maria Jasin
Keyword(s):  
Mammalian Cells ◽  
Atp Hydrolysis ◽  
Chromosomal Rearrangements ◽  
Strand Break ◽  
Dsb Repair ◽  
Mutagenic Potential ◽  
Gross Chromosomal Rearrangements ◽  
Single Strand Annealing ◽  
Chromosomal Breaks ◽  
Strand Annealing

ABSTRACT Repair of chromosomal breaks is essential for cellular viability, but misrepair generates mutations and gross chromosomal rearrangements. We investigated the interrelationship between two homologous-repair pathways, i.e., mutagenic single-strand annealing (SSA) and precise homology-directed repair (HDR). For this, we analyzed the efficiency of repair in mammalian cells in which double-strand break (DSB) repair components were disrupted. We observed an inverse relationship between HDR and SSA when RAD51 or BRCA2 was impaired, i.e., HDR was reduced but SSA was increased. In particular, expression of an ATP-binding mutant of RAD51 led to a >90-fold shift to mutagenic SSA repair. Additionally, we found that expression of an ATP hydrolysis mutant of RAD51 resulted in more extensive gene conversion, which increases genetic loss during HDR. Disruption of two other DSB repair components affected both SSA and HDR, but in opposite directions: SSA and HDR were reduced by mutation of Brca1, which, like Brca2, predisposes to breast cancer, whereas SSA and HDR were increased by Ku70 mutation, which affects nonhomologous end joining. Disruption of the BRCA1-associated protein BARD1 had effects similar to those of mutation of BRCA1. Thus, BRCA1/BARD1 has a role in homologous repair before the branch point of HDR and SSA. Interestingly, we found that Ku70 mutation partially suppresses the homologous-repair defects of BARD1 disruption. We also examined the role of RAD52 in homologous repair. In contrast to yeast, Rad52 − / − mouse cells had no detectable HDR defect, although SSA was decreased. These results imply that the proper genetic interplay of repair factors is essential to limit the mutagenic potential of DSB repair.

scholarly journals Live-cell single-molecule tracking highlights requirements for stable Smc5/6 chromatin association in vivo

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Thomas J Etheridge ◽  
Desiree Villahermosa ◽  
Eduard Campillo-Funollet ◽  
Alex David Herbert ◽  
Anja Irmisch ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Single Molecule ◽  
Chromosomal Rearrangements ◽  
Binding Activity ◽  
Single Molecule Tracking ◽  
Ssdna Binding ◽  
Dna Binding Activity ◽  
Gross Chromosomal Rearrangements ◽  
Genes Encoding

The essential Smc5/6 complex is required in response to replication stress and is best known for ensuring the fidelity of homologous recombination. Using single-molecule tracking in live fission yeast to investigate Smc5/6 chromatin association, we show that Smc5/6 is chromatin associated in unchallenged cells and this depends on the non-SMC protein Nse6. We define a minimum of two Nse6-dependent sub-pathways, one of which requires the BRCT-domain protein Brc1. Using defined mutants in genes encoding the core Smc5/6 complex subunits, we show that the Nse3 double-stranded DNA binding activity and the arginine fingers of the two Smc5/6 ATPase binding sites are critical for chromatin association. Interestingly, disrupting the single-stranded DNA (ssDNA) binding activity at the hinge region does not prevent chromatin association but leads to elevated levels of gross chromosomal rearrangements during replication restart. This is consistent with a downstream function for ssDNA binding in regulating homologous recombination.

scholarly journals Human HELB is a processive motor protein which catalyses RPA clearance from single-stranded DNA

2021 ◽  
Author(s):  
Silvia Hormeno ◽  
Oliver J Wilkinson ◽  
Clara Aicart-Ramos ◽  
Sahiti Kuppa ◽  
Edwin Antony ◽  
...  
Keyword(s):  
Dna Replication ◽  
Direct Observation ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Motor Protein ◽  
Dna Unwinding ◽  
Helicase Activity ◽  
Dna Loops ◽  
Single Stranded Dna ◽  
A Site

Human HELB is a poorly-characterised helicase suggested to play both positive and negative regulatory roles in DNA replication and recombination. In this work, we used bulk and single molecule approaches to characterise the biochemical activities of HELB protein with a particular focus on its interactions with RPA and RPA-ssDNA filaments. HELB is a monomeric protein which binds tightly to ssDNA with a site size of ~20 nucleotides. It couples ATP hydrolysis to translocation along ssDNA in the 5′-to-3′ direction accompanied by the formation of DNA loops and with an efficiency of 1 ATP per base. HELB also displays classical helicase activity but this is very weak in the absence of an assisting force. HELB binds specifically to human RPA which enhances its ATPase and ssDNA translocase activities but inhibits DNA unwinding. Direct observation of HELB on RPA nucleoprotein filaments shows that translocating HELB concomitantly clears RPA from single-stranded DNA.

scholarly journals Inverted DNA Repeats Channel Repair of Distant Double-Strand Breaks into Chromatid Fusions and Chromosomal Rearrangements

2007 ◽  
Vol 27 (7) ◽  
pp. 2601-2614 ◽  
Author(s):  
Kelly VanHulle ◽  
Francene J. Lemoine ◽  
Vidhya Narayanan ◽  
Brandon Downing ◽  
Krista Hull ◽  
...  
Keyword(s):  
Chromosomal Rearrangements ◽  
Inverted Repeats ◽  
Dna Repeats ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Gross Chromosomal Rearrangements ◽  
Double Strand Dna Breaks ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
Break Induced Replication

ABSTRACT Inverted DNA repeats are known to cause genomic instabilities. Here we demonstrate that double-strand DNA breaks (DSBs) introduced a large distance from inverted repeats in the yeast (Saccharomyces cerevisiae) chromosome lead to a burst of genomic instability. Inverted repeats located as far as 21 kb from each other caused chromosome rearrangements in response to a single DSB. We demonstrate that the DSB initiates a pairing interaction between inverted repeats, resulting in the formation of large dicentric inverted dimers. Furthermore, we observed that propagation of cells containing inverted dimers led to gross chromosomal rearrangements, including translocations, truncations, and amplifications. Finally, our data suggest that break-induced replication is responsible for the formation of translocations resulting from anaphase breakage of inverted dimers. We propose a model explaining the formation of inverted dicentric dimers by intermolecular single-strand annealing (SSA) between inverted DNA repeats. According to this model, anaphase breakage of inverted dicentric dimers leads to gross chromosomal rearrangements (GCR). This “SSA-GCR” pathway is likely to be important in the repair of isochromatid breaks resulting from collapsed replication forks, certain types of radiation, or telomere aberrations that mimic isochromatid breaks.

scholarly journals DNA replication machinery prevents Rad52-dependent single-strand annealing that leads to gross chromosomal rearrangements at centromeres

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Atsushi T. Onaka ◽  
Jie Su ◽  
Yasuhiro Katahira ◽  
Crystal Tang ◽  
Faria Zafar ◽  
...  
Keyword(s):  
Dna Replication ◽  
Chromosomal Rearrangements ◽  
Repetitive Sequences ◽  
Single Strand ◽  
Replication Machinery ◽  
Gross Chromosomal Rearrangements ◽  
Single Strand Annealing ◽  
Rad52 Protein ◽  
Strand Annealing ◽  
Recombination Pathway

AbstractHomologous recombination between repetitive sequences can lead to gross chromosomal rearrangements (GCRs). At fission yeast centromeres, Rad51-dependent conservative recombination predominantly occurs between inverted repeats, thereby suppressing formation of isochromosomes whose arms are mirror images. However, it is unclear how GCRs occur in the absence of Rad51 and how GCRs are prevented at centromeres. Here, we show that homology-mediated GCRs occur through Rad52-dependent single-strand annealing (SSA). The rad52-R45K mutation, which impairs SSA activity of Rad52 protein, dramatically reduces isochromosome formation in rad51 deletion cells. A ring-like complex Msh2–Msh3 and a structure-specific endonuclease Mus81 function in the Rad52-dependent GCR pathway. Remarkably, mutations in replication fork components, including DNA polymerase α and Swi1/Tof1/Timeless, change the balance between Rad51-dependent recombination and Rad52-dependent SSA at centromeres, increasing Rad52-dependent SSA that forms isochromosomes. Our results uncover a role of DNA replication machinery in the recombination pathway choice that prevents Rad52-dependent GCRs at centromeres.

scholarly journals Mechanisms underlying genome instability mediated by formation of foldback inversions in Saccharomyces cerevisiae

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Bin-zhong Li ◽  
Christopher D Putnam ◽  
Richard David Kolodner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genome Instability ◽  
Genetic Diseases ◽  
Chromosomal Rearrangements ◽  
Genetic System ◽  
Dna Hairpins ◽  
Large Loop ◽  
Gross Chromosomal Rearrangements ◽  
Single Strand Annealing ◽  
Strand Annealing

Foldback inversions, also called inverted duplications, have been observed in human genetic diseases and cancers. Here, we used a Saccharomyces cerevisiae genetic system that generates gross chromosomal rearrangements (GCRs) mediated by foldback inversions combined with whole-genome sequencing to study their formation. Foldback inversions were mediated by formation of single-stranded DNA hairpins. Two types of hairpins were identified: small-loop hairpins that were suppressed by MRE11, SAE2, SLX1, and YKU80 and large-loop hairpins that were suppressed by YEN1, TEL1, SWR1, and MRC1. Analysis of CRISPR/Cas9-induced double strand breaks (DSBs) revealed that long-stem hairpin-forming sequences could form foldback inversions when proximal or distal to the DSB, whereas short-stem hairpin-forming sequences formed foldback inversions when proximal to the DSB. Finally, we found that foldback inversion GCRs were stabilized by secondary rearrangements, mostly mediated by different homologous recombination mechanisms including single-strand annealing; however, POL32-dependent break-induced replication did not appear to be involved forming secondary rearrangements.

scholarly journals Single-molecule live cell imaging of the Smc5/6 DNA repair complex

2020 ◽  
Author(s):  
Thomas J. Etheridge ◽  
Desiree Villahermosa ◽  
Anja Irmisch ◽  
Adam T. Watson ◽  
Alex Herbert ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Single Molecule ◽  
Chromosomal Rearrangements ◽  
Binding Activity ◽  
Single Molecule Tracking ◽  
Ssdna Binding ◽  
Dna Binding Activity ◽  
Double Stranded Dna ◽  
Gross Chromosomal Rearrangements ◽  
Genes Encoding

AbstractThe Smc5/6 complex is involved in various DNA transactions and is best known for ensuring the fidelity of homologous recombination. We exploit single-molecule tracking in live fission yeast to investigate Smc5/6 chromatin association. We show that Smc5/6 is chromatin associated in unchallenged cells and this depends on the non-SMC protein Nse6. We define a minimum of two Nse6-dependent sub-pathways, one of which requires the BRCT-domain protein Brc1. Using defined mutants in genes encoding the core Smc5/6 complex subunits we show that the Nse3 double-stranded DNA binding activity and the two arginine fingers of the two Smc5/6 ATPase binding sites are critical for chromatin association. Interestingly, disrupting the ssDNA binding activity at the hinge region does not prevent chromatin association. However, unlike a mutant attenuating chromatin association, a mutant that disrupts ssDNA binding results in highly elevated levels of gross chromosomal rearrangements during replication restart. This is consistent with a downstream function for ssDNA binding in regulating homologous recombination.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

scholarly journals Analysis of the Structural Mechanism of ATP Inhibition at the AAA1 Subunit of Cytoplasmic Dynein-1 Using a Chemical “Toolkit”

2021 ◽  
Vol 22 (14) ◽  
pp. 7704
Author(s):  
Sayi’Mone Tati ◽  
Laleh Alisaraie
Keyword(s):  
Binding Affinity ◽  
Atp Hydrolysis ◽  
Critical Role ◽  
Motor Protein ◽  
Structural Data ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Motor Domain ◽  
Conformational Search ◽  
Structural Mechanism

Dynein is a ~1.2 MDa cytoskeletal motor protein that carries organelles via retrograde transport in eukaryotic cells. The motor protein belongs to the ATPase family of proteins associated with diverse cellular activities and plays a critical role in transporting cargoes to the minus end of the microtubules. The motor domain of dynein possesses a hexameric head, where ATP hydrolysis occurs. The presented work analyzes the structure–activity relationship (SAR) of dynapyrazole A and B, as well as ciliobrevin A and D, in their various protonated states and their 46 analogues for their binding in the AAA1 subunit, the leading ATP hydrolytic site of the motor domain. This study exploits in silico methods to look at the analogues’ effects on the functionally essential subsites of the motor domain of dynein 1, since no similar experimental structural data are available. Ciliobrevin and its analogues bind to the ATP motifs of the AAA1, namely, the walker-A (W-A) or P-loop, the walker-B (W-B), and the sensor I and II. Ciliobrevin A shows a better binding affinity than its D analogue. Although the double bond in ciliobrevin A and D was expected to decrease the ligand potency, they show a better affinity to the AAA1 binding site than dynapyrazole A and B, lacking the bond. In addition, protonation of the nitrogen atom in ciliobrevin A and D, as well as dynapyrazole A and B, at the N9 site of ciliobrevin and the N7 of the latter increased their binding affinity. Exploring ciliobrevin A geometrical configuration suggests the E isomer has a superior binding profile over the Z due to binding at the critical ATP motifs. Utilizing the refined structure of the motor domain obtained through protein conformational search in this study exhibits that Arg1852 of the yeast cytoplasmic dynein could involve in the “glutamate switch” mechanism in cytoplasmic dynein 1 in lieu of the conserved Asn in AAA+ protein family.

SINE-B1 Distribution and Chromosome Rearrangements in the South American Proechimys gr. goeldii (Echimyidae, Rodentia)

2021 ◽  
pp. 1-8
Author(s):  
Naiara P. Araújo ◽  
Radarane S. Sena ◽  
Cibele R. Bonvicino ◽  
Gustavo C.S. Kuhn ◽  
Marta Svartman
Keyword(s):  
Transposable Element ◽  
Genome Evolution ◽  
Significant Role ◽  
Sex Chromosomes ◽  
Chromosomal Rearrangements ◽  
Evolutionary Relationship ◽  
Chromosome Rearrangements ◽  
South American ◽  
Gross Chromosomal Rearrangements

<i>Proechimys</i> species are remarkable for their extensive chromosome rearrangements, representing a good model to understand genome evolution. Herein, we cytogenetically analyzed 3 different cytotypes of <i>Proechimys</i> gr. <i>goeldii</i> to assess their evolutionary relationship. We also mapped the transposable element SINE-B1 on the chromosomes of <i>P.</i> gr. <i>goeldii</i> in order to investigate its distribution among individuals and evaluate its possible contribution to karyotype remodeling in this species. SINE-B1 showed a dispersed distribution along chromosome arms and was also detected at the pericentromeric regions of some chromosomes, including pair 1 and the sex chromosomes, which are involved in chromosome rearrangements. In addition, we describe a new cytotype for <i>P.</i> gr. <i>goeldii</i>, reinforcing the significant role of gross chromosomal rearrangements during the evolution of the genus. The results of FISH with SINE-B1 suggest that this issue should be more deeply investigated for a better understanding of its role in the mechanisms involved in the wide variety of <i>Proechimys</i> karyotypes.

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