Search PubMed⌕ Search

Biomedical subjects

C M Radding

Publications and source records attributed to C M Radding.

At least 37 records · Page 2Linked to original sources
PubMed1996

A possible role of the C-terminal domain of the RecA protein. A gateway model for double-stranded DNA binding.

According to the crystal structure, the RecA protein has a domain near the C terminus consisting of amino acid residues 270-328 (from the N terminus). Our model building pointed out the possibility that this domain is a part of "gateway" through which double-stranded DNA finds a path for direct contact with single-stranded DNA within a presynaptic RecA filament in the search for homology. To test this possible function of the domain, we made mutant RecA proteins by site-directed single (or double, in one case) replacement of 2 conserved basic amino acid residues and 5 among 9 nonconserved basic amino acid residues in the domain. Replacement of either of the 2 conserved amino acid residues caused deficiencies in repair of UV-damaged DNA, an in vivo function of RecA protein, whereas the replacement of most (except one) of the tested nonconserved ones gave little or no effect. Purified mutant RecA proteins showed no (or only slight) deficiencies in the formation of presynaptic filaments as assessed by various assays. However, presynaptic filaments of both proteins that had replacement of a conserved amino acid residue had significant defects in binding to and pairing with duplex DNA (secondary binding). These results are consistent with our model that the conserved amino acid residues in the C-terminal domain have a direct role in double-stranded DNA binding and that they constitute a part of a gateway for homologous recognition.

Adenosine Triphosphate↗
PubMed1996

Rad51 expression and localization in B cells carrying out class switch recombination.

Rad51 is a highly conserved eukaryotic homolog of the prokaryotic recombination protein RecA, which has been shown to function in both recombinational repair of DNA damage and meiotic recombination in yeast. In primary murine B cells cultured with lipopolysaccharide (LPS) to stimulate heavy chain class switch recombination, Rad51 protein levels are dramatically induced. Immunofluorescent microscopy shows that anti-Rad51 antibodies stain foci that are localized within the nuclei of switching B cells. Immunohistochemical analysis of splenic sections shows that clusters of cells that stain brightly with anti-Rad51 antibodies are evident within several days after primary immunization and that Rad51 staining in vivo is confined to B cells that are switching from expression of IgM to IgG antibodies. Following switch recombination, B cells populate splenic germinal centers, where somatic hypermutation and clonal proliferation occur. Germinal center B cells are not stained by anti-Rad51 antibodies. Rad51 expression is therefore not coincident with somatic hypermutation, nor does Rad51 expression correlate simply with cell proliferation. These data suggest that Rad51, or a highly related member of the conserved RecA family, may function in class switch recombination.

Amino Acid Sequence↗
PubMed1996

Rec-A protein-mediated irreversible fixation of an oligodeoxyribonucleotide to specific site in DNA.

RecA protein can polymerize on an oligodeoxyribonucleotide to form a filament that finds its homologous sequence in double-stranded DNA. When such an oligonucleotide is linked to psoralen, a photoactivatable DNA intercalator, it irreversibly binds to the homologous site in double stranded DNA as a result of psoralen photoadduct formation at thymidines. The relative efficiency of specific vs. nonspecific binding of an oligonucleotide depended upon the ratio of psoralenated oligonucleotide to total DNA. Na+ ions at concentrations greater than 50 mM eliminated specific binding. Under optimal conditions. the probability of binding of an 80-mer oligonucleotide to a specific site was > 10(5) times greater than that of binding to any single nonspecific site. Under the conditions described, RecA-mediated photoadduction was equally efficient with superhelical and linear double-stranded DNA.

Base Sequence↗
PubMed1996

Mammalian ubiquitin-conjugating enzyme Ubc9 interacts with Rad51 recombination protein and localizes in synaptonemal complexes.

Hsubc9, a human gene encoding a ubiquitin-conjugating enzyme, has been cloned. The 18-kDa HsUbc9 protein is homologous to the ubiquitin-conjugating enzymes Hus5 of Schizosaccharomyces pombe and Ubc9 of Saccharomyces cerevisiae. The Hsubc9 gene complements a ubc9 mutation of S. cerevisiae. It has been mapped to chromosome 16p13.3 and is expressed in many human tissues, with the highest levels in testis and thymus. According to the Ga14 two-hybrid system analysis, HsUbc9 protein interacts with human recombination protein Rad51. A mouse homolog, Mmubc9, encodes an amino acid sequence that is identical to the human protein. In mouse spermatocytes, MmUbc9 protein, like Rad51 protein, localizes in synaptonemal complexes, which suggests that Ubc9 protein plays a regulatory role in meiosis.

Amino Acid Sequence↗
PubMed1995

Uptake and processing of duplex DNA by RecA nucleoprotein filaments: insights provided by a mixed population of dynamic and static intermediates.

In the polarized strand exchange that is promoted by Escherichia coli RecA protein, when the initiating end of a duplex DNA molecule is blocked by heterology, the homologous distal end nonetheless forms a joint with single-stranded DNA, but strand exchange in that joint cannot be completed because the strand that would otherwise be displaced lacks a free 5' end. Instead, 2/3 to 3/4 of such distal joints cyclically form and dissociate. Dissociation requires the hydrolysis of ATP (Burnett et al., 1994). Observations on DNase protection revealed that consistent with their dynamic nature, these joints were heterogeneous in length, extending from the labeled distal end of the duplex up to 600 base pairs within the homologous region. Switching of base pairs was undetectable in this fraction of distal joints. However, the other 1/3 to 1/4 of distal joints, which did not cycle, were as long as the entire homologous region (6 kb), and underwent complete switching of base pairs. The formation of these static joints occurred at a rate in excess of 100 bp per second, without requiring hydrolysis of ATP. These and earlier observations suggest that the RecA filament containing single-stranded DNA rapidly incorporates duplex DNA into a coaxial three-stranded helix by a passive process, whereas additional energy is required to convert the three-stranded intermediate into products or back into substrates, both of which involve the unwinding of many turns of three-stranded helix.

Adenosine Triphosphate↗
PubMed1995

Nuclear foci of mammalian Rad51 recombination protein in somatic cells after DNA damage and its localization in synaptonemal complexes.

Rad51 protein of Saccharomyces cerevisiae is a structural homolog of the Escherichia coli recombination enzyme RecA. In yeast, the Rad51 protein is required for mitotic and meiotic recombination and for repair of double-strand breaks in DNA. We have used antibodies raised against the homologous human protein, HsRad51, expressed in E. coli, to visualize the spatial distribution of the protein in mammalian somatic and meiotic cells. In cultured human cells, the HsRad51 protein is concentrated in multiple discrete foci in the nucleoplasm; it is largely absent from cytoplasm and nucleoli. After treatment of cells with methyl methanesulfonate, ultraviolet irradiation, or 137Cs irradiation, the percentage of cells with HsRad51 protein immunofluorescence increases; the same cells show unscheduled DNA synthesis. Induction of Rad51 foci is blocked by inhibitors of transcription. In mouse pachytene spermatocytes, the mouse homolog of Rad51 protein is highly enriched in synaptonemal complexes that are formed between the paired homologous chromosomes during meiotic prophase. We conclude that the mammalian proteins homologous to yeast Rad51 are involved in repair of DNA damage and recombinational repair during meiosis.

Animals↗
PubMed1995

RecA protein mediates homologous recognition via non-Watson-Crick bonds in base triplets.

E. coli RecA protein, the prototype of a class, forms a helical nucleoprotein filament on single-stranded DNA that recognizes homology in duplex DNA, and initiates the exchange of strands in homologous recombination. The discovery of this reaction some years ago posed a quandary on how a third strand recognizes homology in duplex DNA, whose Watson-Crick bonds face inward in a hydrophobic core of stacked bases. Recent studies have shown that RecA protein promotes homologous recognition via non-Watson-Crick bonds in base triplets. The intermediates in the RecA reaction differ distinctly from triplex DNA that forms non-enzymically. The biological significance of the novel set of DNA interactions by which RecA protein effects homologous recognition is indicated by the importance of this protein in recombination, and the widespread distribution of homologous proteins in prokaryotes and eukaryotes.

Base Composition↗
PubMed1995

How specific is the first recognition step of homologous recombination?

The Escherichia coli RecA protein promotes homologous recognition in base triplets via non-Watson-Crick bonds that differ from those formed nonenzymically from DNA consisting of runs of purines or pyrimidines. Base substitutions reveal recognition to be permissive, consistent with a search for homology that achieves speed at the cost of precision.

Binding Sites↗
PubMed1994

Joints made by RecA protein in the interior of linear duplex DNA: effects of single-stranded ends, length of homology, and dynamic state.

The importance of 3' single-stranded ends in homologous recombination led us to reevaluate reactions of single strands at homologous sites in the interior of linear duplex DNA. As the length of homology increased, the yield of joints increased up to about 2 kb of homology, at which the apparent yields were the same at either 3' or 5' single-stranded ends, or away from ends, although such joints were qualitatively different. In the presence of RecA protein and ATP, joints that formed between any of these single strands and interior sequences in duplex DNA were in a dynamic state in which they constantly recycled. Consequently, their apparent yields at steady state were sensitive to conditions of reaction, such as the concentrations of DNA, salt, and ATP vs ADP, observations which rationalize conflicting reports in the literature. The dynamic state of joints in the interior of duplex DNA strengthens the conclusion [see Burnett et al. (1994)] that RecA protein dissociates joints when it cannot displace the 5' end of a strand from the recipient duplex.

Adenosine Diphosphate↗
PubMed1994

A chimeric Rec-A protein that implicates non-Watson-Crick interactions in homologous pairing.

The helical filament formed by RecA protein on single-stranded DNA plays an important role in homologous recombination and pairs with a complementary single strand or homologous duplex DNA. The RecA nucleoprotein filament also recognizes an identical single strand. The chimeric protein, RecAc38, forms a nucleoprotein filament that recognizes a complementary strand but is defective in recognition of duplex DNA, and is associated with phenotypic defects in repair and recombination. As described here, RecAc38 nucleoprotein filament is also defective in recognition of an identical strand, either when the filament has within it a single strand or duplex DNA. A model that postulates three DNA binding sites rationalizes these observations and suggests that the third binding site mediates non-Watson-Crick interactions that are instrumental in recognition of homology in duplex DNA.

Base Sequence↗
PubMed1994

Formation of base triplets by non-Watson-Crick bonds mediates homologous recognition in RecA recombination filaments.

Whereas complementary strands of DNA recognize one another by forming Watson-Crick base pairs, the way in which RecA protein enables a single strand to recognize homology in duplex DNA has remained unknown. Recent experiments, however, have shown that a single plus strand in the RecA filament can recognize an identical plus strand via bonds that, by definition, are non-Watson-Crick. In experiments reported here, base substitutions had the same qualitative and quantitative effects on the pairing of two identical strands in the RecA filament as on the recognition of duplex DNA by a third strand, indicating that similar non-Watson-Crick interactions govern both reactions.

Base Composition↗
PubMed1994

Resolution of the three-stranded recombination intermediate made by RecA protein. An essential role of ATP hydrolysis.

Previous work has shown that triplex DNA is an intermediate in homologous pairing and strand exchange promoted by RecA protein. Heterology at the proximal end of duplex DNA blocks strand exchange, but triplex joints form nonetheless at the homologous distal end. Experiments on the formation and processing of distal joints revealed that the yield of distal joints depends critically on the concentration of RecA-coated single strands and the adequacy of the ATP-regeneration system, and reflects a steady state. Distal joints reversibly formed and dissociated, as shown by several methods, including a chase with unlabeled duplex DNA. Controls excluded a contribution of exonucleolytic nibbling to the formation of distal joints and the stability of the deproteinized product. RecA protein was bound preferentially by putative triplex sites both in isolated proximal and distal joints. These high affinity sites disappeared from proximal joints as strand exchange progressed, and disappeared from distal joints as the joints dissociated. Dissociation of distal joints under all conditions, however, was completely arrested by the addition of ATP gamma S. Distal triplex joints can be as long as six kilobases. The observed inhibition of the dissociation of such long non-productive triplex intermediates by ATP gamma S leads us to propose that an essential role of ATP hydrolysis in RecA recombinational exchanges may be to ensure that no potentially troublesome triplex DNA remains in the cell.

Adenosine Triphosphate↗
PubMed1993

Interactions of three strands in joints made by RecA protein.

RecA protein from Escherichia coli has been used to form a triple-stranded DNA structure from either single-stranded M13 DNA or a single-stranded oligonucleotide plus a duplex oligonucleotide with a hairpin loop. The secondary structure of purified deproteinized triplex was examined by probing with DNase I, P1 nuclease, potassium permanganate, and diethyl pyrocarbonate. The two strands destined to form heteroduplex DNA showed the same patterns of chemical modification and enzymatic digestion as control duplex DNA, indicating that they formed a normal duplex substructure. However, the nascent outgoing strand showed properties consistent with a novel triplex structure: most of its purine residues, especially adenines, were hyperreactive to all probes. The patterns of digestion by DNase I and P1 nuclease indicated that the nascent outgoing strand was not a freely mobile or single-stranded branch but rather was still interacting with the newly formed heteroduplex DNA. On the basis of the planar base triads proposed previously (Rao et al., 1993) and energy minimization of a third strand in the major groove of B-form DNA, we derived a model that helps to rationalize the properties revealed by chemical and enzymatic probing.

Bacteriophage M13↗
PubMed1993

Inhibition of RNA polymerase II transcription by oligonucleotide-RecA protein filaments targeted to promoter sequences.

In the presence of RecA protein, which plays a major role in genetic recombination in Escherichia coli, an oligodeoxyribonucleotide can find its homologous counterpart in double-stranded DNA and form triple-stranded structures. A triple-stranded structure formed by an oligonucleotide with a sequence overlapping essential regulatory elements of a viral promoter, such as TATA or GC boxes, inhibited in vitro transcription driven by RNA polymerase II. An oligonucleotide with eight nucleotides homologous to its target suppressed RNA polymerase II activity in HeLa cell extracts. This procedure offers a potential alternative to the usual mutational analysis of transcriptional promoters.

Base Sequence↗
PubMed1993

Homologous recognition promoted by RecA protein via non-Watson-Crick bonds between identical DNA strands.

The RecA protein of Escherichia coli forms a nucleoprotein filament that promotes homologous recognition and subsequent strand exchange between a single strand and duplex DNA via a three-stranded intermediate. Recognition of homology within three-stranded nucleoprotein complexes, which is probably central to genetic recombination, is not well understood as compared with the mutual recognition of complementary single strands by Watson-Crick base pairing. Using oligonucleotides, we examined the determinants of homologous recognition within RecA nucleoprotein filaments. Filaments that contained a single strand of DNA recognized homology not only in a complementary oligonucleotide but also in an identical oligonucleotide, whether their respective sugar-phosphate backbones were antiparallel or parallel, and a filament that contained duplex DNA showed the same polymorphic versatility in the recognition of homology. Recognition of self by a filament that contains a single strand reveals that RecA filaments can recognize homology via non-Watson-Crick hydrogen bonds. Recognition of multiple forms of the same sequence by duplex DNA in the filament shows that it primarily senses base-sequence homology, and suggests that recognition can be accomplished prior to the establishment of new Watson-Crick base pairs in heteroduplex products. However, unlike the initial recognition of homology, strand exchange is stereospecific, requiring the proper antiparallel orientation of complementary strands.

Bacteriophage M13↗
PubMed1993

Homologous recognition and triplex formation promoted by RecA protein between duplex oligonucleotides and single-stranded DNA.

RecA protein formed a stable triplex from a 33 bp duplex oligonucleotide and a circular plus strand of M13 DNA when a hairpin connection at the proximal end of the homologous duplex oligonucleotide blocked displacement of the 5' end of its own plus strand. An oligonucleotide with a hairpin connection at the other end yielded five times fewer joints that survived deproteinization, and an ordinary duplex oligonucleotide yielded none. The stability of the three-stranded structure was not attributable to exonucleolytic nibbling of the 3' end of the hairpin oligonucleotide, which could generate a region of stable duplex DNA. In the triplexes, the hairpin duplex became more accessible to copper phenanthroline, exhibited novel sites of cleavage by DNase I, and resisted digestion by Escherichia coli exonuclease I. The enzymatic methylation of only two residues at N-6 adenine and two at N-4 cytosine in the hairpin duplex prior to the pairing reaction lowered the tm of triplexes by 8 deg.C, whereas extensive methylation at N-7 guanine by dimethyl sulfate had no effect. These results are discussed in relation to possible models of triplex DNA.

Adenine↗
PubMed1993

Ribosomal protein S1 and NusA protein complexed to recombination protein beta of phage lambda.

The red genes of bacteriophage lambda specify two proteins, exonuclease and beta protein, which are essential for general recombination of lambda in recA cells. Earlier studies suggested that these proteins form an equimolar complex (C. M. Radding, J. Rosenweig, F. Richards, and E. Cassuto, J. Biol. Chem. 246:2510-2512, 1971). A more recent study indicated that beta protein forms a strong complex with an unknown polypeptide of 70 kDa (K. Muniyappa and C. M. Radding, J. Biol. Chem. 261:7472-7478, 1986). In the present study, in addition to the complex of beta and the 70-kDa protein, a new association of beta protein with a 65-kDa protein was observed. N-terminal sequencing identified these proteins as host-encoded ribosomal protein S1 and transcription terminator protein NusA.

Amino Acid Sequence↗