scholarly journals ATP-dependent and independent functions of Rad54 in genome maintenance

2011 ◽  
Vol 192 (5) ◽  
pp. 735-750 ◽  
Author(s):  
Sheba Agarwal ◽  
Wiggert A. van Cappellen ◽  
Aude Guénolé ◽  
Berina Eppink ◽  
Sam E.V. Linsen ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Protein A ◽  
Dna Double Strand Breaks ◽  
Genome Maintenance ◽  
Focus Formation ◽  
Recombination Proteins ◽  
Real Time Analysis ◽  
Independent Functions

Rad54, a member of the SWI/SNF protein family of DNA-dependent ATPases, repairs DNA double-strand breaks (DSBs) through homologous recombination. Here we demonstrate that Rad54 is required for the timely accumulation of the homologous recombination proteins Rad51 and Brca2 at DSBs. Because replication protein A and Nbs1 accumulation is not affected by Rad54 depletion, Rad54 is downstream of DSB resection. Rad54-mediated Rad51 accumulation does not require Rad54’s ATPase activity. Thus, our experiments demonstrate that SWI/SNF proteins may have functions independent of their ATPase activity. However, quantitative real-time analysis of Rad54 focus formation indicates that Rad54’s ATPase activity is required for the disassociation of Rad54 from DNA and Rad54 turnover at DSBs. Although the non–DNA-bound fraction of Rad54 reversibly interacts with a focus, independent of its ATPase status, the DNA-bound fraction is immobilized in the absence of ATP hydrolysis by Rad54. Finally, we show that ATP hydrolysis by Rad54 is required for the redistribution of DSB repair sites within the nucleus.

scholarly journals Weaving DNA strands: structural insight on ATP hydrolysis in RecA-induced homologous recombination

2019 ◽  
Vol 47 (15) ◽  
pp. 7798-7808
Author(s):  
Benjamin Boyer ◽  
Claudia Danilowicz ◽  
Mara Prentiss ◽  
Chantal Prévost
Keyword(s):  
Homologous Recombination ◽  
Atp Hydrolysis ◽  
Strand Exchange ◽  
Geometrical Approach ◽  
Structural Level ◽  
Dna Double Strand Breaks ◽  
Sequence Recognition ◽  
Dna Strands ◽  
Reverse Strand ◽  
Dna Strand

Abstract Homologous recombination is a fundamental process in all living organisms that allows the faithful repair of DNA double strand breaks, through the exchange of DNA strands between homologous regions of the genome. Results of three decades of investigation and recent fruitful observations have unveiled key elements of the reaction mechanism, which proceeds along nucleofilaments of recombinase proteins of the RecA family. Yet, one essential aspect of homologous recombination has largely been overlooked when deciphering the mechanism: while ATP is hydrolyzed in large quantity during the process, how exactly hydrolysis influences the DNA strand exchange reaction at the structural level remains to be elucidated. In this study, we build on a previous geometrical approach that studied the RecA filament variability without bound DNA to examine the putative implication of ATP hydrolysis on the structure, position, and interactions of up to three DNA strands within the RecA nucleofilament. Simulation results on modeled intermediates in the ATP cycle bring important clues about how local distortions in the DNA strand geometries resulting from ATP hydrolysis can aid sequence recognition by promoting local melting of already formed DNA heteroduplex and transient reverse strand exchange in a weaving type of mechanism.

scholarly journals Regulatory control of Sgs1 and Dna2 during eukaryotic DNA end resection

2019 ◽  
Vol 116 (13) ◽  
pp. 6091-6100 ◽  
Author(s):  
Chaoyou Xue ◽  
Weibin Wang ◽  
J. Brooks Crickard ◽  
Corentin J. Moevus ◽  
Youngho Kwon ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Single Molecule ◽  
Protein A ◽  
Regulatory Control ◽  
Dna Double Strand Breaks ◽  
Nucleosome Remodeling ◽  
Damage Repair ◽  
Eukaryotic Dna ◽  
Dna End Resection ◽  
Dna Ends

In the repair of DNA double-strand breaks by homologous recombination, the DNA break ends must first be processed into 3′ single-strand DNA overhangs. In budding yeast, end processing requires the helicase Sgs1 (BLM in humans), the nuclease/helicase Dna2, Top3-Rmi1, and replication protein A (RPA). Here, we use single-molecule imaging to visualize Sgs1-dependent end processing in real-time. We show that Sgs1 is recruited to DNA ends through Top3-Rmi1–dependent or –independent means, and in both cases Sgs1 is maintained in an immoble state at the DNA ends. Importantly, the addition of Dna2 triggers processive Sgs1 translocation, but DNA resection only occurs when RPA is also present. We also demonstrate that the Sgs1–Dna2–Top3-Rmi1–RPA ensemble can efficiently disrupt nucleosomes, and that Sgs1 itself possesses nucleosome remodeling activity. Together, these results shed light on the regulatory interplay among conserved protein factors that mediate the nucleolytic processing of DNA ends in preparation for homologous recombination-mediated chromosome damage repair.

scholarly journals Cellular Deficiency of Werner Syndrome Protein or RECQ1 Promotes Genotoxic Potential of Hydroquinone and Benzo[a]pyrene Exposure

2014 ◽  
Vol 33 (5) ◽  
pp. 373-381 ◽  
Author(s):  
Mamatha Garige ◽  
Sudha Sharma
Keyword(s):  
Werner Syndrome ◽  
Protein A ◽  
Protein Kinase Activity ◽  
Environmental Toxicants ◽  
Dna Double Strand Breaks ◽  
Genome Maintenance ◽  
Werner Syndrome Protein ◽  
Carcinogenic Effects ◽  
Dependent Protein ◽  
Syndrome Protein

The 5 known RecQ helicases in humans (RECQ1, BLM, WRN, RECQL4, and RECQ5) have demonstrated roles in diverse genome maintenance mechanisms but their functions in safeguarding the genome from environmental toxicants are poorly understood. Here, we have evaluated a potential role of WRN (mutated in Werner syndrome) and RECQ1 (the most abundant homolog of WRN) in hydroquinone (HQ)- and benzo[a]pyrene (BaP)-induced genotoxicity. Silencing of WRN or RECQ1 expression in HeLa cells increased their sensitivity to HQ and BaP but elicited distinct DNA damage response. The RECQ1-depleted cells exhibited increased replication protein A phosphorylation, Chk1 activation, and DNA double-strand breaks (DSBs) as compared to control or WRN-depleted cells following exposure to BaP treatment. The BaP-induced DSBs in RECQ1-depleted cells were dependent on DNA-dependent protein kinase activity. Notably, loss of WRN in RECQ1-depleted cells ameliorated BaP toxicity. Collectively, our results provide first indication of nonredundant participation of WRN and RECQ1 in protection from the potentially carcinogenic effects of BaP and HQ.

scholarly journals Recombination and chromosome segregation

2004 ◽  
Vol 359 (1441) ◽  
pp. 61-69 ◽  
Author(s):  
David J. Sherratt ◽  
Britta Søballe ◽  
François–Xavier Barre ◽  
Sergio Filipe ◽  
Ivy Lau ◽  
...  
Keyword(s):  
Cell Division ◽  
Homologous Recombination ◽  
Chromosome Segregation ◽  
Atp Hydrolysis ◽  
Daughter Cells ◽  
Recombination Proteins ◽  
Recombination System ◽  
Associated Proteins ◽  
Stalled Replication Forks ◽  
Circular Chromosomes

The duplication of DNA and faithful segregation of newly replicated chromosomes at cell division is frequently dependent on recombinational processes. The rebuilding of broken or stalled replication forks is universally dependent on homologous recombination proteins. In bacteria with circular chromosomes, crossing over by homologous recombination can generate dimeric chromosomes, which cannot be segregated to daughter cells unless they are converted to monomers before cell division by the conserved Xer site–specific recombination system. Dimer resolution also requires FtsK, a division septum–located protein, which coordinates chromosome segregation with cell division, and uses the energy of ATP hydrolysis to activate the dimer resolution reaction. FtsK can also translocate DNA, facilitate synapsis of sister chromosomes and minimize entanglement and catenation of newly replicated sister chromosomes. The visualization of the replication/recombination–associated proteins, RecQ and RarA, and specific genes within living Escherichia coli cells, reveals further aspects of the processes that link replication with recombination, chromosome segregation and cell division, and provides new insight into how these may be coordinated.

scholarly journals Smc5/6 Is Required for Repair at Collapsed Replication Forks

2006 ◽  
Vol 26 (24) ◽  
pp. 9387-9401 ◽  
Author(s):  
Eleni Ampatzidou ◽  
Anja Irmisch ◽  
Matthew J. O'Connell ◽  
Johanne M. Murray
Keyword(s):  
Homologous Recombination ◽  
Protein Complexes ◽  
S Phase ◽  
Replication Forks ◽  
Replication Arrest ◽  
Recombination Proteins ◽  
Independent Functions ◽  
Chromatid Cohesion ◽  
Recombination Repair ◽  
Structural Maintenance Of Chromosome

ABSTRACT In eukaryotes, three pairs of structural-maintenance-of-chromosome (SMC) proteins are found in conserved multisubunit protein complexes required for chromosomal organization. Cohesin, the Smc1/3 complex, mediates sister chromatid cohesion while two condensin complexes containing Smc2/4 facilitate chromosome condensation. Smc5/6 scaffolds an essential complex required for homologous recombination repair. We have examined the response of smc6 mutants to the inhibition of DNA replication. We define homologous recombination-dependent and -independent functions for Smc6 during replication inhibition and provide evidence for a Rad60-independent function within S phase, in addition to a Rad60-dependent function following S phase. Both genetic and physical data show that when forks collapse (i.e., are not stabilized by the Cds1Chk2 checkpoint), Smc6 is required for the effective repair of resulting lesions but not for the recruitment of recombination proteins. We further demonstrate that when the Rad60-dependent, post-S-phase Smc6 function is compromised, the resulting recombination-dependent DNA intermediates that accumulate following release from replication arrest are not recognized by the G2/M checkpoint.

scholarly journals Single-molecule visualization of human RECQ5 interactions with single-stranded DNA recombination intermediates

2020 ◽  
Author(s):  
Chaoyou Xue ◽  
Lucia Molnarova ◽  
Justin B Steinfeld ◽  
Weixing Zhao ◽  
Chujian Ma ◽  
...  
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Protein A ◽  
Dna Recombination ◽  
Binding Activity ◽  
Negative Regulator ◽  
Genome Maintenance ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Translocation Velocity

Abstract RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51–ssDNA filaments. RECQ5 interacts with RAD51 through protein–protein contacts, and disruption of this interface through a RECQ5–F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51–K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51–I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.

scholarly journals Human Rad54 Protein Stimulates DNA Strand Exchange Activity of hRad51 Protein in the Presence of Ca2+

2004 ◽  
Vol 279 (50) ◽  
pp. 52042-52051 ◽  
Author(s):  
Olga M. Mazina ◽  
Alexander V. Mazin
Keyword(s):  
Homologous Recombination ◽  
Atp Hydrolysis ◽  
Specific Interaction ◽  
Strand Exchange ◽  
Rad51 Protein ◽  
Recombination Proteins ◽  
Dna Strand Exchange ◽  
Dna Strand ◽  
Exchange Activity

Rad51 and Rad54 proteins play a key role in homologous recombination in eukaryotes. Recently, we reported that Ca2+is requiredin vitrofor human Rad51 protein to form an active nucleoprotein filament that is important for the search of homologous DNA and for DNA strand exchange, two critical steps of homologous recombination. Here we find that Ca2+is also required for hRad54 protein to effectively stimulate DNA strand exchange activity of hRad51 protein. This finding identifies Ca2+as a universal cofactor of DNA strand exchange promoted by mammalian homologous recombination proteinsin vitro. We further investigated the hRad54-dependent stimulation of DNA strand exchange. The mechanism of stimulation appeared to include specific interaction of hRad54 protein with the hRad51 nucleoprotein filament. Our results show that hRad54 protein significantly stimulates homology-independent coaggregation of dsDNA with the filament, which represents an essential step of the search for homologous DNA. The results obtained indicate that hRad54 protein serves as a dsDNA gateway for the hRad51-ssDNA filament, promoting binding and an ATP hydrolysis-dependent translocation of dsDNA during the search for homologous sequences.

scholarly journals A Proximity Ligation Method to Detect Proteins Bound to Single-Stranded DNA after DNA End Resection at DNA Double-Strand Breaks

2021 ◽  
Vol 5 (1) ◽  
pp. 3
Author(s):  
Faith C. Fowler ◽  
Jessica K. Tyler
Keyword(s):  
Homologous Recombination ◽  
Protein A ◽  
Replication Protein A ◽  
Strand Break ◽  
Replication Protein ◽  
Proximity Ligation Assay ◽  
Dna Double Strand Breaks ◽  
Color Channel ◽  
Single Stranded Dna ◽  
Proximity Ligation

After a DNA double-strand break, cells utilize either non-homologous end joining or homologous recombination to repair the broken DNA ends. Homologous recombination requires extensive nucleolytic processing of one of the DNA strands, resulting in long stretches of 3′ single-strand DNA overhangs. Typically, single-stranded DNA is measured using immunofluorescence microscopy to image the foci of replication protein A, a single-stranded DNA-binding protein. Microscopy analysis of bromodeoxyuridine foci under nondenaturing conditions has also been used to measure single-stranded DNA. Here, we describe a proximity ligation assay which uses genome-wide bromodeoxyuridine incorporation to label single-stranded DNA in order to measure the association of a protein of interest with single-stranded DNA. This method is advantageous over traditional foci analysis because it is more direct and specific than traditional foci co-localization microscopy methods, uses only one color channel, and can reveal protein-single-stranded DNA interactions that are rare and potentially undetectable using traditional microscopy methods. We show here the association of replication protein A and bromodeoxyuridine as proof-of-concept.

RPA phosphorylation facilitates RAD52 dependent homologous recombination in BRCA-deficient cells

2021 ◽  
Author(s):  
Alison C. Carley ◽  
Manisha Jalan ◽  
Shyamal Subramanyam ◽  
Rohini Roy ◽  
Gloria E.O. Borgstahl ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Protein A ◽  
Double Strand Breaks ◽  
Strand Exchange ◽  
Dna Duplexes ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Synthetically Lethal ◽  
Mutant Cells

Loss of RAD52 is synthetically lethal in BRCA-deficient cells, owing to its role in backup homologous recombination (HR) repair of DNA double-strand breaks (DSBs). In HR in mammalian cells, DSBs are processed to single-stranded DNA (ssDNA) overhangs, which are then bound by Replication Protein A(RPA). RPA is exchanged for RAD51 by mediator proteins: in mammals BRCA2 is the primary mediator, however, RAD52 provides an alternative mediator pathway in BRCA-deficient cells. RAD51 stimulates strand exchange between homologous DNA duplexes, a critical step in HR. RPA phosphorylation and de-phosphorylation are important for HR, but its effect on RAD52 mediator function is unknown. Here, we show that RPA phosphorylation is required for RAD52 to salvage HR in BRCA-deficient cells. Using BRCA2-depleted human cells, in which the only available mediator pathway is RAD52-dependent, the expression of phosphorylation-deficient RPA mutant reduced HR. Furthermore, RPA-phospho-mutant cells showed reduced association of RAD52 with RAD51. Interestingly, there was no effect of RPA phosphorylation on RAD52 recruitment to repair foci. Finally, we show that RPA phosphorylation does not affect RAD52-dependent ssDNA annealing. Thus, although RAD52 can be recruited independently of RPA’s phosphorylation status, RPA phosphorylation is required for RAD52’s association with RAD51, and its subsequent promotion of RAD52-mediated HR.

Interdomain regulation of the ATPase activity of the ABC transporter haemolysin B from Escherichia coli

2016 ◽  
Vol 473 (16) ◽  
pp. 2471-2483 ◽  
Author(s):  
Sven Reimann ◽  
Gereon Poschmann ◽  
Kerstin Kanonenberg ◽  
Kai Stühler ◽  
Sander H.J. Smits ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Abc Transporter ◽  
Biochemical Characterization ◽  
Atp Hydrolysis ◽  
Protein A ◽  
Maximum Velocity ◽  
Hydrolytic Activity ◽  
Secretion Systems ◽  
Hydrolytic Activities ◽  
Wide Range

Type 1 secretion systems (T1SS) transport a wide range of substrates across both membranes of Gram-negative bacteria and are composed of an outer membrane protein, a membrane fusion protein and an ABC (ATP-binding cassette) transporter. The ABC transporter HlyB (haemolysin B) is part of a T1SS catalysing the export of the toxin HlyA in E. coli. HlyB consists of the canonical transmembrane and nucleotide-binding domains. Additionally, HlyB contains an N-terminal CLD (C39-peptidase-like domain) that interacts with the transport substrate, but its functional relevance is still not precisely defined. In the present paper, we describe the purification and biochemical characterization of detergent-solubilized HlyB in the presence of its transport substrate. Our results exhibit a positive co-operativity in ATP hydrolysis. We characterized further the influence of the CLD on kinetic parameters by using an HlyB variant lacking the CLD (HlyB∆CLD). The biochemical parameters of HlyB∆CLD revealed an increased basal maximum velocity but no change in substrate-binding affinity in comparison with full-length HlyB. We also assigned a distinct interaction of the CLD and a transport substrate (HlyA1), leading to an inhibition of HlyB hydrolytic activity at low HlyA1 concentrations. At higher HlyA1 concentrations, we observed a stimulation of the hydrolytic activities of both HlyB and HlyB∆CLD, which was completely independent of the interaction of HlyA1 with the CLD. Notably, all observed effects on ATPase activity, which were also analysed in detail by mass spectrometry, were independent of the HlyA1 secretion signal. These results assign an interdomain regulatory role for the CLD modulating the hydrolytic activity of HlyB.

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