Biomedical subjects

S C Kowalczykowski

Publications and source records attributed to S C Kowalczykowski.

At least 37 records · Page 2Linked to original sources

PubMed1999

A single mutation, RecB(D1080A,) eliminates RecA protein loading but not Chi recognition by RecBCD enzyme.

Homologous recombination and double-stranded DNA break repair in Escherichia coli are initiated by the multifunctional RecBCD enzyme. After binding to a double-stranded DNA end, the RecBCD enzyme unwinds and degrades the DNA processively. This processing is regulated by the recombination hot spot, Chi (chi: 5'-GCTGGTGG-3'), which induces a switch in the polarity of DNA degradation and activates RecBCD enzyme to coordinate the loading of the DNA strand exchange protein, RecA, onto the single-stranded DNA products of unwinding. Recently, a single mutation in RecB, Asp-1080 --> Ala, was shown to create an enzyme (RecB(D1080A)CD) that is a processive helicase but not a nuclease. Here we show that the RecB(D1080A)CD enzyme is also unable to coordinate the loading of the RecA protein, regardless of whether chi sites are present in the DNA. However, the RecB(D1080A)CD enzyme does respond to chi sites by inactivating in a chi-dependent manner. These data define a locus of the RecBCD enzyme that is essential not only for nuclease function but also for the coordination of RecA protein loading.

DNA Helicases↗

PubMed1999

A novel property of the RecA nucleoprotein filament: activation of double- stranded DNA for strand exchange in trans.

RecA protein catalyzes DNA strand exchange, a basic step of homologous recombination. Upon binding to single-stranded DNA (ssDNA), RecA protein forms a helical nucleoprotein filament. Normally, this nucleoprotein filament binds double-stranded DNA (dsDNA) and promotes exchange of base pairs between this dsDNA and the homologous ssDNA that is contained within this filament. Here, we demonstrate that this bound dsDNA can be activated by interaction with a heterologous RecA nucleoprotein filament for a novel type of strand exchange with homologous ssDNA that is external to, and, therefore, not within, the filament. We refer to this novel DNA strand exchange as being in trans. Thus, the RecA nucleoprotein filament is a protein scaffold that activates dsDNA for strand exchange with ssDNA either within the filament or external to it. This new property demonstrates that the RecA nucleoprotein filament makes dsDNA receptive for DNA strand exchange, and it defines an early step of the homology recognition mechanism.

Adenosine Triphosphate↗

PubMed1999

The RecBC enzyme loads RecA protein onto ssDNA asymmetrically and independently of chi, resulting in constitutive recombination activation.

Double-strand DNA break repair and homologous recombination in Escherichia coli proceed by the RecBCD pathway, which is regulated by cis-acting elements known as chi sites. A crucial feature of this regulation is the RecBCD enzyme-directed loading of RecA protein specifically onto the 3'-terminal, chi-containing DNA strand. Here we show that RecBC enzyme (lacking the RecD subunit) loads RecA protein constitutively onto the 3'-terminal DNA strand, with no requirement for chi. This strand is preferentially utilized in homologous pairing reactions. We propose that RecA protein loading is a latent property of the RecBCD holoenzyme, which is normally blocked by the RecD subunit and is revealed following interaction with chi.

Blotting, Southern↗

PubMed1999

The simultaneous binding of two double-stranded DNA molecules by Escherichia coli RecA protein.

We have characterized the double-stranded DNA (dsDNA) binding properties of RecA protein, using an assay based on changes in the fluorescence of 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complexes. Here we use fluorescence, nitrocellulose filter-binding, and DNase I-sensitivity assays to demonstrate the binding of two duplex DNA molecules by the RecA protein filament. We previously established that the binding stoichiometry for the RecA protein-dsDNA complex is three base-pairs per RecA protein monomer, in the presence of ATP. In the presence of ATPgammaS, however, the binding stoichiometry depends on the MgCl2 concentration. The stoichiometry is 3 bp per monomer at low MgCl2 concentrations, but changes to 6 bp per monomer at higher MgCl2 concentrations, with the transition occurring at approximately 5 mM MgCl2. Above this MgCl2 concentration, the dsDNA within the RecA nucleoprotein complex becomes uncharacteristically sensitive to DNase I digestion. For these reasons we suggest that, at the elevated MgCl2 conditions, the RecA-dsDNA nucleoprotein filament can bind a second equivalent of dsDNA. These results demonstrate that RecA protein has the capacity to bind two dsDNA molecules, and they suggest that RecA or RecA-like proteins may effect homologous recognition between intact DNA duplexes.

Adenosine Triphosphate↗

PubMed1999

The DNA binding properties of Saccharomyces cerevisiae Rad51 protein.

Saccharomyces cerevisiae Rad51 protein is the paradigm for eukaryotic ATP-dependent DNA strand exchange proteins. To explain some of the unique characteristics of DNA strand exchange promoted by Rad51 protein, when compared with its prokaryotic homologue the Escherichia coli RecA protein, we analyzed the DNA binding properties of the Rad51 protein. Rad51 protein binds both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in an ATP- and Mg2+-dependent manner, over a wide range of pH, with an apparent binding stoichiometry of approximately 1 protein monomer per 4 (+/-1) nucleotides or base pairs, respectively. Only dATP and adenosine 5'-gamma-(thiotriphosphate) (ATPgammaS) can substitute for ATP, but binding in the presence of ATPgammaS requires more than a 5-fold stoichiometric excess of protein. Without nucleotide cofactor, Rad51 protein binds both ssDNA and dsDNA but only at pH values lower than 6.8; in this case, the apparent binding stoichiometry covers the range of 1 protein monomer per 6-9 nucleotides or base pairs. Therefore, Rad51 protein displays two distinct modes of DNA binding. These binding modes are not inter-convertible; however, their initial selection is governed by ATP binding. On the basis of these DNA binding properties, we conclude that the main reason for the low efficiency of the DNA strand exchange promoted by Rad51 protein in vitro is its enhanced dsDNA-binding ability, which inhibits both the presynaptic and synaptic phases of the DNA strand exchange reaction as follows: during presynapsis, Rad51 protein interacts with and stabilizes secondary structures in ssDNA thereby inhibiting formation of a contiguous nucleoprotein filament; during synapsis, Rad51 protein inactivates the homologous dsDNA partner by directly binding to it.

Adenosine Triphosphate↗

PubMed1999

RecQ helicase and topoisomerase III comprise a novel DNA strand passage function: a conserved mechanism for control of DNA recombination.

E. coli RecQ protein is a multifunctional helicase with homologs that include the S. cerevisiae Sgs1 helicase and the H. sapiens Wrn and Blm helicases. Here we show that RecQ helicase unwinds a covalently closed double-stranded DNA (dsDNA) substrate and that this activity specifically stimulates E. coli topoisomerase III (Topo III) to fully catenate dsDNA molecules. We propose that these proteins functionally interact and that their shared activity is responsible for control of DNA recombination. RecQ helicase has a comparable effect on the Topo III homolog of S. cerevisiae, consistent with other RecQ and Topo III homologs acting together in a similar capacity. These findings highlight a novel, conserved activity that offers insight into the function of the other RecQ-like helicases.

Adenosine Triphosphatases↗

PubMed1999

Enhanced monomer-monomer interactions can suppress the recombination deficiency of the recA142 allele.

The RecA142 protein, in which valine is substituted for isoleucine-225, is defective for genetic recombination in vivo and for DNA strand exchange activity in vitro under conventional growth and reaction conditions respectively. However, we show that mildly acidic conditions restore both the in vitro DNA strand exchange activity and the in vivo function of RecA142 protein, suggesting that recombination function can be restored by a slight change in protein structure elicited by protonation. Indeed, we identified an intragenic suppressor of the recombination deficiency of the recA142 allele. This suppressor mutation is a substitution of leucine for glutamine at position 124. Based on the three-dimensional structure, the Q-124L substitution is predicted to make a new monomer-monomer contact with residue phenylalanine-21 of the adjacent RecA monomer. The Q-124L mutation is not allele specific, because it also suppresses the recombination deficiency of a recA deletion (Delta9), lacking nine amino acids at the amino-terminus, presumably by reinforcing the monomer-monomer interactions that are attenuated by the Delta9 deletion. Expression of RecA(Q-124L) protein is toxic to Escherichia coli, presumably because of enhanced affinity for DNA. We speculate as to how enhanced monomer-monomer interactions and acidic pH conditions can restore the recombination activity of some defective recA alleles.

Acids↗

PubMed1998

Reconstitution of an SOS response pathway: derepression of transcription in response to DNA breaks.

E. coli responds to DNA damage by derepressing the transcription of about 20 genes that make up the SOS pathway. Genetic analyses have shown that SOS induction in response to double-stranded DNA (dsDNA) breaks requires LexA repressor, and the RecA and RecBCD enzymes--proteins best known for their role as initiators of dsDNA break repair and homologous recombination. Here we demonstrate that purified RecA protein, RecBCD enzyme, single-stranded DNA-binding (SSB) protein, and LexA repressor respond to dsDNA breaks in vitro by derepressing transcription from an SOS promoter. Interestingly, derepression is more rapid if the DNA containing the dsDNA break has a chi recombination hot spot (5'-GCTGGTGG-3'), suggesting a novel regulatory role for one of the most overrepresented octamers in the E. coli genome.

Bacterial Proteins↗

PubMed1998

Modulation of RecA nucleoprotein function by the mutagenic UmuD'C protein complex.

The RecA, UmuC, and UmuD' proteins are essential for error-prone, replicative bypass of DNA lesions. Normally, RecA protein mediates homologous pairing of DNA. We show that purified Umu(D')2C blocks this recombination function. Biosensor measurements establish that the mutagenic complex binds to the RecA nucleoprotein filament with a stoichiometry of one Umu(D')2C complex for every two RecA monomers. Furthermore, Umu(D')2C competitively inhibits LexA repressor cleavage but not ATPase activity, implying that Umu(D')2C binds in or proximal to the helical groove of the RecA nucleoprotein filament. This binding reduces joint molecule formation and even more severely impedes DNA heteroduplex formation by RecA protein, ultimately blocking all DNA pairing activity and thereby abridging participation in recombination function. Thus, Umu(D')2C restricts the activities of the RecA nucleoprotein filament and presumably, in this manner, recruits it for mutagenic repair function. This modulation by Umu(D')2C is envisioned as a key event in the transition from a normal mode of genomic maintenance by "error-free" recombinational repair, to one of "error-prone" DNA replication.

Bacterial Proteins↗

PubMed1998

SSB protein controls RecBCD enzyme nuclease activity during unwinding: a new role for looped intermediates.

The RecBCD enzyme of Escherichia coli initiates homologous recombination by unwinding and simultaneously degrading DNA from a double-stranded DNA end. Single-stranded DNA loops are intermediates of this unwinding process. Here we show that SSB protein reduces the level of DNA degradation by RecBCD enzyme during unwinding, by binding to these ssDNA intermediates. Prior to interaction with the recombination hot spot chi, RecBCD enzyme has both 3'-->5' exonuclease and a weaker 5'-->3' exonuclease activity. We show that degradation of the 5'-terminal strand at the entry site is much more extensive in the absence of SSB protein. After interaction with chi, the level of 5'-->3' exonuclease activity is increased; as expected, degradation of the 5'-strand is also elevated in the absence of SSB protein. Furthermore, we show that, in the absence of SSB protein, the RecBCD enzyme is inhibited by the ssDNA products of unwinding; SSB protein alleviates this inhibition. These results provide insight into the organization of helicase and nuclease domains within the RecBCD enzyme, and also suggest a new level at which the nuclease activity of RecBCD enzyme is controlled. Hence, they offer new insight into the role of SSB protein in the initiation phase of recombination.

DNA Helicases↗

PubMed1998

The reduced levels of chi recognition exhibited by the RecBC1004D enzyme reflect its recombination defect in vivo.

Homologous recombination in Escherichia coli is initiated by the RecBCD enzyme and is stimulated by an 8-nucleotide element known as Chi (chi). We present a detailed biochemical characterization of a mutant RecBCD enzyme, designated RecBC1004D, that displays a reduced level of chi site recognition. Initially characterized genetically as unable to respond to the chi sequence, we provide evidence to indicate that the ability of this mutant enzyme to respond to chi is reduced rather than lost; the mutant displays about 20-fold lower chi recognition than wild-type RecBCD enzyme. Although this enzyme exhibits wild-type levels of double-stranded DNA exonuclease, helicase, and ATPase activity, its ability to degrade single-stranded DNA is enhanced 2-3-fold. The data presented here suggest that the reduced recombination proficiency of the recBC1004D strain observed in vivo results from a basal level of modification of the RecBC1004D enzyme at both chi-specific, as well as nonspecific, DNA sequences.

Adenosine Triphosphatases↗

PubMed1998

DNA strand exchange proteins: a biochemical and physical comparison.

Homologous genetic recombination is an essential biological process that involves the pairing and exchange of DNA between two homologous chromosomes or DNA molecules. It is of fundamental importance to the preservation of genomic integrity, the production of genetic diversity, and the proper segregation of chromosomes. In Escherichia coli, the RecA protein is essential to recombination, and biochemical analysis demonstrates that it is responsible for the crucial steps of homologous pairing and DNA strand exchange. The presence of RecA-like proteins, or their functional equivalents, in bacteriophage, other eubacteria, archaea, and eukaryotes, confirms that the mechanism of homologous pairing and DNA strand exchange is conserved throughout all forms of life. This review focuses on the biochemical and physical characteristics of DNA strand exchange proteins from three diverse organisms: RecA protein from E. coli, UvsX protein from Bacteriophage T4, and RAD51 protein from Saccharomyces cerevisiae.

Adenosine Triphosphate↗

PubMed1998

DNA annealing by RAD52 protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA.

Homologous recombination in Saccharomyces cerevisiae depends critically on RAD52 function. In vitro, Rad52 protein preferentially binds single-stranded DNA (ssDNA), mediates annealing of complementary ssDNA, and stimulates Rad51 protein-mediated DNA strand exchange. Replication protein A (RPA) is a ssDNA-binding protein that is also crucial to the recombination process. Herein we report that Rad52 protein effects the annealing of RPA-ssDNA complexes, complexes that are otherwise unable to anneal. The ability of Rad52 protein to promote annealing depends on both the type of ssDNA substrate and ssDNA binding protein. RPA allows, but slows, Rad52 protein-mediated annealing of oligonucleotides. In contrast, RPA is almost essential for annealing of longer plasmid-sized DNA but has little effect on the annealing of poly(dT) and poly(dA), which are relatively long DNA molecules free of secondary structure. These results suggest that one role of RPA in Rad52 protein-mediated annealing is the elimination of DNA secondary structure. However, neither Escherichia coli ssDNA binding protein nor human RPA can substitute in this reaction, indicating that RPA has a second role in this process, a role that requires specific RPA-Rad52 protein interactions. This idea is confirmed by the finding that RPA, which is complexed with nonhomologous ssDNA, inhibits annealing but the human RPA-ssDNA complex does not. Finally, we present a model for the early steps of the repair of double-strand DNA breaks in yeast.

DNA Repair↗

PubMed1998

RadA protein is an archaeal RecA protein homolog that catalyzes DNA strand exchange.

With the discovery that the Saccharomyces cerevisiae Rad51 protein is both structurally and functionally similar to the Escherichia coli RecA protein, the RecA paradigm for homologous recombination was extended to the Eucarya. The ubiquitous presence of RecA and Rad51 protein homologs raises the question of whether this archetypal protein exists within the third domain of life, the Archaea. Here we present the isolation of a Rad51/RecA protein homolog from the archaeon Sulfolobus solfataricus, and show that this protein, RadA, possesses the characteristics of a DNA strand exchange protein: The RadA protein is a DNA-dependent ATPase, forms a nucleoprotein filament on DNA, and catalyzes DNA pairing and strand exchange.

Adenosine Triphosphatases↗

PubMed1998

RecQ helicase, in concert with RecA and SSB proteins, initiates and disrupts DNA recombination.

RecQ helicase is important to homologous recombination and DNA repair in Escherichia coli. We demonstrate that RecQ helicase, in conjunction with RecA and SSB proteins, can initiate recombination events in vitro. In addition, RecQ protein is capable of unwinding a wide variety of DNA substrates, including joint molecules formed by RecA protein. These data are consistent with RecQ helicase assuming two roles in the cell; it can be (1) an initiator of homologous recombination, or (2) a disrupter of joint molecules formed by aberrant recombination. These findings also shed light on the function of the eukaryotic homologs of RecQ helicase, the Sgs1, Blm, and Wrn helicases.

Adenosine Triphosphatases↗

PubMed1998

The function of the secondary DNA-binding site of RecA protein during DNA strand exchange.

RecA protein features two distinct DNA-binding sites. During DNA strand exchange, the primary site binds to single-stranded DNA (ssDNA), forming the helical RecA nucleoprotein filament. The weaker secondary site binds double-stranded DNA (dsDNA) during the homology search process. Here we demonstrate that this site has a second important function. It binds the ssDNA strand that is displaced from homologous duplex DNA during DNA strand exchange, stabilizing the initial heteroduplex DNA product. Although the high affinity of the secondary site for ssDNA is essential for DNA strand exchange, it renders DNA strand exchange sensitive to an excess of ssDNA which competes with dsDNA for binding. We further demonstrate that single-stranded DNA-binding protein can sequester ssDNA, preventing its binding to the secondary site and thereby assisting at two levels: it averts the inhibition caused by an excess of ssDNA and prevents the reversal of DNA strand exchange by removing the displaced strand from the secondary site.

Binding Sites↗

PubMed1998

Rad52 protein stimulates DNA strand exchange by Rad51 and replication protein A.

The generation of a double-strand break in the Saccharomyces cerevisiae genome is a potentially catastrophic event that can induce cell-cycle arrest or ultimately result in loss of cell viability. The repair of such lesions is strongly dependent on proteins encoded by the RAD52 epistasis group of genes (RAD50-55, RAD57, MRE11, XRS2), as well as the RFA1 and RAD59 genes. rad52 mutants exhibit the most severe phenotypic defects in double-strand break repair, but almost nothing is known about the biochemical role of Rad52 protein. Rad51 protein promotes DNA strand exchange and acts similarly to RecA protein. Yeast Rad52 protein interacts with Rad51 protein, binds single-stranded DNA and stimulates annealing of complementary single-stranded DNA. We find that Rad52 protein stimulates DNA strand exchange by targeting Rad51 protein to a complex of replication protein A (RPA) with single-stranded DNA. Rad52 protein affects an early step in the reaction, presynaptic filament formation, by overcoming the inhibitory effects of the competitor, RPA. Furthermore, stimulation is dependent on the concerted action of both Rad51 protein and RPA, implying that specific protein-protein interactions between Rad52 protein, Rad51 protein and RPA are required.

DNA, Single-Stranded↗

PubMed1998

Binding of double-stranded DNA by Escherichia coli RecA protein monitored by a fluorescent dye displacement assay.

We have developed a new assay to characterize the double-stranded DNA (dsDNA) binding properties of RecA protein. This assay is based on measurement of changes in the fluorescence of a 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complex upon RecA protein binding. The binding of RecA protein to a complex of DAPI and dsDNA results in displacement of the bound DAPI, producing a decrease in the observed fluorescence. DAPI displacement is dependent on both RecA protein and ATP; dATP and, to a lesser extent, UTP and dCTP also support the DAPI displacement reaction, but dGTP, GTP, dITP and TTP do not. Binding stoichiometry for the RecA protein-dsDNA complex measured by DAPI displacement is 3 bp per RecA protein monomer in the presence of ATP. These results, taken together with data for mutant RecA proteins, suggest that this DAPI displacement assay monitors formation of the high affinity DNA binding state of RecA protein. Since this state of RecA protein defines the form of the nucleoprotein filament that is active in DNA strand exchange, these findings raise the possibility that the RecA protein-dsDNA filament may possess a homologous pairing capacity.

Adenosine Triphosphate↗