How strand exchange protein function benefits from ATP hydrolysis

Molecular motors perform essential functions in the cell and have the potential to provide insights into the basis of many important processes. A unique property of molecular motors is their ability to convert energy from ATP hydrolysis into work, enabling the motors to bind to and move along cytoskeletal filaments. The mechanism of energy conversion by molecular motors is not yet understood and may lead to the discovery of new biophysical principles. Mutant analysis could provide valuable information, but it is not obvious how to obtain mutants that are informative for study. The analysis presented here points out several strategies for obtaining mutants by selection from molecular or genetic screens, or by rational design. Mutants that are expected to provide important information about the motor mechanism include ATPase mutants, which interfere with the nucleotide hydrolysis cycle, and uncoupling mutants, which unlink basic motor activities and reveal their interdependence. Natural variants can also be exploited to provide unexpected information about motor function. This general approach to uncovering protein function by analysis of informative mutants is applicable not only to molecular motors, but to other proteins of interest.

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Rad51 catalytic mutants differentially affect the Rad51 nucleoprotein filament in vivo

10.1101/070276 ◽

2016 ◽

Author(s):

Maureen M. Mundia ◽

Alissa C. Magwood ◽

Mark D. Baker

Keyword(s):

Homologous Recombination ◽

Dna Synthesis ◽

Cell Lines ◽

Atp Hydrolysis ◽

Dominant Negative ◽

Strand Exchange ◽

Wild Type ◽

Hybridoma Cell Lines ◽

Insight Into

ABSTRACTIn this study, we utilized mouse hybridoma cell lines stably expressing ectopic wild-type Rad51, or the Rad51-K133A and Rad51-K133R catalytic mutants deficient in ATP binding and ATP hydrolysis, respectively, to investigate effects on the Rad51 nucleoprotein filament in vivo. Immunoprecipitation studies reveal interactions between ectopic wild-type Rad51, Rad51-K133A and Rad51-K133R and endogenous Rad51, Brca2 and p53 proteins. Importantly, the expression of Rad51-K133A and Rad51-K133R catalytic mutants (but not wild-type Rad51) targets endogenous Rad51, Brca2 and p53 proteins for proteasome-mediated degradation. Expression of Rad51-K133R significantly reduces nascent DNA synthesis (3’ polymerization) during homologous recombination (HR), but the effects of Rad51-K133A on 3’ polymerization are considerably more severe. Provision of additional wild-type Rad51 in cell lines expressing Rad51-K133A or Rad51-K133R does not restore diminished levels of endogenous Brca2, Rad51 or p53, nor restore the deficiency in 3’ polymerization. Cells expressing Rad51-K133A are also significantly reduced in their capacity to drive strand exchange through regions of heterology. Our results reveal an interesting mechanistic dichotomy in the way mutant Rad51-K133A and Rad51-K133R proteins influence 3’ polymerization and provide novel insight into the mechanism of their dominant-negative phenotypes.

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Characterization of an ATP-dependent DNA strand transferase from human cells.

Molecular and Cellular Biology ◽

10.1128/mcb.7.9.3124 ◽

1987 ◽

Vol 7 (9) ◽

pp. 3124-3130 ◽

Cited By ~ 33

Author(s):

D Ganea ◽

P Moore ◽

L Chekuri ◽

R Kucherlapati

Keyword(s):

Dna Sequences ◽

Atp Hydrolysis ◽

Reca Protein ◽

Strand Exchange ◽

Cell Nuclei ◽

In Vitro Recombination ◽

Dna Strand ◽

Exchange Activity

We have characterized an enzymatic activity from human cell nuclei which is capable of catalyzing strand exchange between homologous DNA sequences. The strand exchange activity was Mg2+ dependent and required ATP hydrolysis. In addition, it was capable of promoting reannealing of homologous DNA sequences and could form nucleoprotein networks in a fashion reminiscent of purified bacterial RecA protein. Using an in vitro recombination assay, we also showed that the strand exchange activity was biologically important. The factor(s) responsible for the activity has been partially purified.

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Molecular and genetic analysis of the gene encoding the Saccharomyces cerevisiae strand exchange protein Sep1

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2593-2608.1991 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2593-2608 ◽

Cited By ~ 1

Author(s):

D X Tishkoff ◽

A W Johnson ◽

R D Kolodner

Keyword(s):

Saccharomyces Cerevisiae ◽

Open Reading Frame ◽

Strand Exchange ◽

Wild Type ◽

Homologous Pairing ◽

Gene Encoding ◽

Reading Frame ◽

Cloned Gene ◽

Diploid Cells ◽

Exchange Protein

Vegetatively grown Saccharomyces cerevisiae cells contain an activity that promotes a number of homologous pairing reactions. A major portion of this activity is due to strand exchange protein 1 (Sep1), which was originally purified as a 132,000-Mr species (R. Kolodner, D. H. Evans, and P. T. Morrison, Proc. Natl. Acad. Sci. USA 84:5560-5564, 1987). The gene encoding Sep1 was cloned, and analysis of the cloned gene revealed a 4,587-bp open reading frame capable of encoding a 175,000-Mr protein. The protein encoded by this open reading frame was overproduced and purified and had a relative molecular weight of approximately 160,000. The 160,000-Mr protein was at least as active in promoting homologous pairing as the original 132,000-Mr species, which has been shown to be a fragment of the intact 160,000-Mr Sep1 protein. The SEP1 gene mapped to chromosome VII within 20 kbp of RAD54. Three Tn10LUK insertion mutations in the SEP1 gene were characterized. sep1 mutants grew more slowly than wild-type cells, showed a two- to fivefold decrease in the rate of spontaneous mitotic recombination between his4 heteroalleles, and were delayed in their ability to return to growth after UV or gamma irradiation. Sporulation of sep1/sep1 diploids was defective, as indicated by both a 10- to 40-fold reduction in spore formation and reduced spore viability of approximately 50%. The majority of sep1/sep1 diploid cells arrested in meiosis after commitment to recombination but prior to the meiosis I cell division. Return-to-growth experiments showed that sep1/sep1 his4X/his4B diploids exhibited a five- to sixfold greater meiotic induction of His+ recombinants than did isogenic SEP1/SEP1 strains. sep1/sep1 mutants also showed an increased frequency of exchange between HIS4, LEU2, and MAT and a lack of positive interference between these markers compared with wild-type controls. The interaction between sep1, rad50, and spo13 mutations suggested that SEP1 acts in meiosis in a pathway that is parallel to the RAD50 pathway.

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Long-range intramolecular allostery and regulation in the dynein-like AAA protein Mdn1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2002792117 ◽

2020 ◽

Vol 117 (31) ◽

pp. 18459-18469

Author(s):

Keith J. Mickolajczyk ◽

Paul Dominic B. Olinares ◽

Yiming Niu ◽

Nan Chen ◽

Sara E. Warrington ◽

...

Keyword(s):

Electron Microscopy ◽

Mass Spectrometry ◽

Protein Function ◽

Atp Hydrolysis ◽

Native Mass Spectrometry ◽

Chemical Probes ◽

Rich Region ◽

Aaa Atpase ◽

Aaa Protein ◽

Bimolecular Interaction

Mdn1 is an essential mechanoenzyme that uses the energy from ATP hydrolysis to physically reshape and remodel, and thus mature, the 60S subunit of the ribosome. This massive (>500 kDa) protein has an N-terminal AAA (ATPase associated with diverse cellular activities) ring, which, like dynein, has six ATPase sites. The AAA ring is followed by large (>2,000 aa) linking domains that include an ∼500-aa disordered (D/E-rich) region, and a C-terminal substrate-binding MIDAS domain. Recent models suggest that intramolecular docking of the MIDAS domain onto the AAA ring is required for Mdn1 to transmit force to its ribosomal substrates, but it is not currently understood what role the linking domains play, or why tethering the MIDAS domain to the AAA ring is required for protein function. Here, we use chemical probes, single-particle electron microscopy, and native mass spectrometry to study the AAA and MIDAS domains separately or in combination. We find that Mdn1 lacking the D/E-rich and MIDAS domains retains ATP and chemical probe binding activities. Free MIDAS domain can bind to the AAA ring of this construct in a stereo-specific bimolecular interaction, and, interestingly, this binding reduces ATPase activity. Whereas intramolecular MIDAS docking appears to require a treatment with a chemical inhibitor or preribosome binding, bimolecular MIDAS docking does not. Hence, tethering the MIDAS domain to the AAA ring serves to prevent, rather than promote, MIDAS docking in the absence of inducing signals.

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RecA Protein from the Extremely Radioresistant Bacterium Deinococcus radiodurans: Expression, Purification, and Characterization

Journal of Bacteriology ◽

10.1128/jb.184.6.1649-1660.2002 ◽

2002 ◽

Vol 184 (6) ◽

pp. 1649-1660 ◽

Cited By ~ 51

Author(s):

Jong-Il Kim ◽

Ajay K. Sharma ◽

Stephen N. Abbott ◽

Elizabeth A. Wood ◽

David W. Dwyer ◽

...

Keyword(s):

Deinococcus Radiodurans ◽

Atp Hydrolysis ◽

Reca Protein ◽

Strand Exchange ◽

Exchange Reactions ◽

Ssdna Binding ◽

E Coli ◽

Dna Strand Exchange ◽

Ssdna Binding Protein ◽

Dna Strand

ABSTRACT The RecA protein of Deinococcus radiodurans (RecADr) is essential for the extreme radiation resistance of this organism. The RecADr protein has been cloned and expressed in Escherichia coli and purified from this host. In some respects, the RecADr protein and the E. coli RecA (RecAEc) proteins are close functional homologues. RecADr forms filaments on single-stranded DNA (ssDNA) that are similar to those formed by the RecAEc. The RecADr protein hydrolyzes ATP and dATP and promotes DNA strand exchange reactions. DNA strand exchange is greatly facilitated by the E. coli SSB protein. As is the case with the E. coli RecA protein, the use of dATP as a cofactor permits more facile displacement of bound SSB protein from ssDNA. However, there are important differences as well. The RecADr protein promotes ATP- and dATP-dependent reactions with distinctly different pH profiles. Although dATP is hydrolyzed at approximately the same rate at pHs 7.5 and 8.1, dATP supports an efficient DNA strand exchange only at pH 8.1. At both pHs, ATP supports efficient DNA strand exchange through heterologous insertions but dATP does not. Thus, dATP enhances the binding of RecADr protein to ssDNA and the displacement of ssDNA binding protein, but the hydrolysis of dATP is poorly coupled to DNA strand exchange. The RecADr protein thus may offer new insights into the role of ATP hydrolysis in the DNA strand exchange reactions promoted by the bacterial RecA proteins. In addition, the RecADr protein binds much better to duplex DNA than the RecAEc protein, binding preferentially to double-stranded DNA (dsDNA) even when ssDNA is present in the solutions. This may be of significance in the pathways for dsDNA break repair in Deinococcus.

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

Nucleic Acids Research ◽

10.1093/nar/gkz667 ◽

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.

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Molecular and genetic analysis of the gene encoding the Saccharomyces cerevisiae strand exchange protein Sep1.

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2593 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2593-2608 ◽

Cited By ~ 63

Author(s):

D X Tishkoff ◽

A W Johnson ◽

R D Kolodner

Keyword(s):

Saccharomyces Cerevisiae ◽

Open Reading Frame ◽

Strand Exchange ◽

Wild Type ◽

Homologous Pairing ◽

Gene Encoding ◽

Reading Frame ◽

Cloned Gene ◽

Diploid Cells ◽

Exchange Protein

Vegetatively grown Saccharomyces cerevisiae cells contain an activity that promotes a number of homologous pairing reactions. A major portion of this activity is due to strand exchange protein 1 (Sep1), which was originally purified as a 132,000-Mr species (R. Kolodner, D. H. Evans, and P. T. Morrison, Proc. Natl. Acad. Sci. USA 84:5560-5564, 1987). The gene encoding Sep1 was cloned, and analysis of the cloned gene revealed a 4,587-bp open reading frame capable of encoding a 175,000-Mr protein. The protein encoded by this open reading frame was overproduced and purified and had a relative molecular weight of approximately 160,000. The 160,000-Mr protein was at least as active in promoting homologous pairing as the original 132,000-Mr species, which has been shown to be a fragment of the intact 160,000-Mr Sep1 protein. The SEP1 gene mapped to chromosome VII within 20 kbp of RAD54. Three Tn10LUK insertion mutations in the SEP1 gene were characterized. sep1 mutants grew more slowly than wild-type cells, showed a two- to fivefold decrease in the rate of spontaneous mitotic recombination between his4 heteroalleles, and were delayed in their ability to return to growth after UV or gamma irradiation. Sporulation of sep1/sep1 diploids was defective, as indicated by both a 10- to 40-fold reduction in spore formation and reduced spore viability of approximately 50%. The majority of sep1/sep1 diploid cells arrested in meiosis after commitment to recombination but prior to the meiosis I cell division. Return-to-growth experiments showed that sep1/sep1 his4X/his4B diploids exhibited a five- to sixfold greater meiotic induction of His+ recombinants than did isogenic SEP1/SEP1 strains. sep1/sep1 mutants also showed an increased frequency of exchange between HIS4, LEU2, and MAT and a lack of positive interference between these markers compared with wild-type controls. The interaction between sep1, rad50, and spo13 mutations suggested that SEP1 acts in meiosis in a pathway that is parallel to the RAD50 pathway.

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