Charles M. Radding

STUDIES ON THE SYNTHESIS AND SECRETION OF SERUM LIPOPROTEINS BY RAT LIVER SLICES

There is at present a great deal of interest in factors determining the levels of serum lipids because of their possible relation to atherosclerosis. Since the serum lipids circulate only in the form of lipid-protein complexes, the mechanism of production of these complexes and their delivery into and removal from the serum must be understood before there is a sound basis for defining the homeostatic mechanisms controlling the levels of individual lipid components. While factors influe7ncing the metabolism of any one of the individual lipid moieties may influence the serum levels of that moiety, such an effect must ultimately be expressed indirectly through an alteration in the rate or pattern of metabolism of the lipid-protein complexes of which it is a part. The present experiments were undertaken in order to define an in vitro system in which lipoprotein biosynthesis could be studied without the many complicating variables encountered in whole-animal studies. It is generally accepted that the liver is the principal source of the serum lipids (1-3) and it has been assumed, but not proved, that the liver is also the source of the protein moiety of the serum lipoproteins. Wehave previously described the incorporation by perfused rat liver and by rat liver slices of labeled amino acids into proteins with densities similar to those of the serum lipoproteins (4, 5). The studies reported here provide evidence that the high density lipoproteins synthesized and secreted by rat liver slices are identical with the high density lipoproteins of normal rat serum. The time course of synthesis of both the protein and the lipid moieties in the total lipoprotein fraction, the rate of appearance of radioactive lipoprotein in the medium, and the net changes in protein and lipid in liver slice and medium are presented. The effects of cholesterol

Homologous pairing in genetic recombination: recA protein makes joint molecules of gapped circular DNA and closed circular DNA

The recA protein, which is essential for genetic recombination in E. coli, promotes the homologous pairing of double-stranded DNA and linear single-stranded DNA, thereby forming a three-stranded joint molecule called a D loop. Single-stranded DNA stimulates recA protein to unwind double-stranded DNA. By a presumably related mechanism, recA protein promoted the homologous pairing of two circular double-stranded molecules when one of them has a gap in one strand. The two molecules were joined at homologous sites by noncovalent bonds. The covalently closed molecule remained intact and was not topologically linked to the intact circular strand of the gapped substrate. Electron microscopy showed that molecules were usually linked at two or more nearby points. The junctions in most molecules were shorter than 300 nucleotides. Sometimes the region between two extreme points was separated into two arms, producing an ellipsoidal loop (called an eye loop). The junctions in these biparental joint molecules were frequently remote from the site of the gap. We infer that a free end of the interrupted strand crosseover to form a structure like a D loop which moved away from the gap by branch migration.

Uptake of homologous single-stranded fragments by superhelical DNA: II. Characterization of the reaction

Abstract We have studied the association of superhelical DNA (RFI) ‡ of phage G4 with defined single-stranded fragments isolated after cleavage of viral (+) strands by endonuclease R · Hae III. The sedimentation rates of complexes formed by uptake of different single-stranded restriction fragments by G4 RFI were consistent with the view that base-pairing between the two components causes unwinding of superhelical turns, with one negative superhelical turn removed for every ten nucleotide residues of third strand taken up. The combining ratio of superhelical DNA and a single specific fragment was close to unity. At high concentrations of salt, nitrocellulose filters efficiently retained complexes of superhelical DNA and homologous fragments, which provided the basis for a rapid assay, and permitted the estimation of the thermodynamic and kinetic parameters of strand uptake in vitro . The reaction is reversible, with an apparent K eq of approximately 10 6 m −1 . Apparent rate constants, k 1 , for uptake of different fragments (85 to 1100 nucleotides long) varied about fourfold, with no obvious relationship to the length of the fragment. In 10 m m -Tris · HCl (pH 7.5), 200 m m -NaCl, fragments were taken up most rapidly at about 75 °C. Under these conditions, the apparent k 1 for a fragment 250 nucleotides long was approximately 600 m −1 s −1 , which is two or three orders of magnitude slower than the calculated rate of association of complementary strands of that length. At physiological temperatures, appreciable rates of strand uptake were seen only at low concentrations of salt (4 m m -Na + in 10 m m -Tris · HCl), and were one or two orders of magnitude less than the rate at 75 °C in 200 m m -NaCl. At a given concentration of counterion a threshold temperature exists above which the rate of reaction rises sharply from an undetectable level. Thermodynamic calculations indicate that the reaction is entropically driven, and that the rate is limited by a step exhibiting a positive entropy and enthalpy of activation. The data are consistent with a model for strand uptake in which the rate-limiting step is the unstacking of a small number of base-pairs in the superhelical DNA. Stabilization and extension of the nucleus of base-pairs formed with the incoming strand is favored by the decrease in free energy associated with removal of superhelical turns.

Insertions, deletions and mismatches in heteroduplex DNA made by recA protein

E. coli recA protein promotes the pairing of circular single strands with linear duplex DNA and the subsequent formation of large heteroduplex joints. From fd and M13 DNA, recA protein can make heteroduplex joints that include every kind of single base-pair mismatch. Aided by E. coli single-strand binding protein (SSB) and ATP regeneration, recA protein can incorporate into heteroduplex DNA insertions that are hundreds of base pairs long, whether the extra DNA is located initially in either the single-stranded or the double-stranded substrate. The ability of recA protein to span large insertions in the duplex DNA indicates that it unwinds a sizeable number of turns in advance of the growing heteroduplex joint. These observations show that an enzymatic basis exists in E. coli for forming extensively mismatched heteroduplex DNA, which might be involved in conversion-like events associated with recombination.

The topology of homologous pairing promoted by RecA protein

In addition to catalyzing the pairing of linear single-stranded DNA with homologous duplex DNA, recA protein promotes the pairing of circular single strands with linear duplex DNA or nicked circular duplex DNA, and of gapped circular duplex DNA with superhelical DNA. RecA protein will thus produce joint molecules of DNA at a high frequency from a pair of homologous molecules if one of them is single-stranded or partially single-stranded, and if either one has a free end. The structure made from a linear single strand and duplex DNA is a D loop. The joint molecule made from circular single-stranded DNA and linear duplex DNA is a branched structure in which the circular strand has displaced a strand from one end of the duplex molecule. In these structures, the heteroduplex regions reach sizes approaching that of full-length fd DNA. When we used restriction fragments of duplex fd DNA that were approximately half-length, we found circular molecules that were half duplex and half single-stranded. Similarly, single-stranded circles displaced a strand from nicked circular duplex DNA, yielding structures related to those made with linear duplex DNA, as well as other structures. Our observations indicate that purified recA protein catalyzes a concerted strand transfer with several features of particular biological interest, including the initiation of a strand crossover (in some cases perhaps the crossing back of a strand as well) and the production of long heteroduplex joints by a kind of branch migration. While a free end permits interwinding of DNA strands and the formation of joints containing stable right-handed helices, the free end is not essential for the promotion of homologous pairing by recA protein. When we mixed phage G4 double-stranded DNA and recA protein with single-stranded circular M13 DNA containing an insert of 274 bases of G4 DNA, we observed by electron microscopy the formation of a few percent of complexes in which single-stranded circular DNA and duplex DNA were joined side by side in the region of shared sequence homology. The frequency of such complexes was twenty to thirty times greater than that observed in a control mixture of G4 duplex DNA and single-stranded circular fd DNA, molecules which do not share a region of extensive homology. We conclude that recA protein can promote homologous association of a single strand and duplex DNA without the plectonemic colling that characterizes the normal Watson-Crick structure of DNA.

Visualization of RecA protein and its association with DNA: A priming effect of single-strand-binding protein

A stoichiometric interaction of RecA protein with single-stranded DNA promotes homologous pairing of the single strand with duplex DNA and subsequent polar formation of a heteroduplex joint. Escherichia coli single-strand-binding (SSB) protein augments these reactions. Electron microscopic observations suggest structural bases for these interactions. Without triphosphates or DNA, RecA protein forms short linear filaments. With added circular single-stranded DNA, it forms extended circular filaments as well as collapsed and aggregated complexes of protein and DNA. The extended circular filaments are stiff and regular in appearance, contrasting with the convoluted structure formed by SSB protein and single-stranded DNA. Together, these two proteins form mixed filaments, which mostly resemble the extended structures containing RecA protein; moreover, SSB protein accelerates formation of extended filaments more than 50-fold, increasing the yield of these structures at the expense of heterogeneous aggregates. Other observations further define the interactions of RecA protein with partially single-stranded DNA, and the effects of ATP gamma S on the tendency of RecA protein to form polymeric structures even in the absence of DNA.

Synapsis and the formation of paranemic joints by E. coli RecA protein

E. coli RecA protein promotes the homologous pairing of a single strand with duplex DNA even when certain features of the substrates, such as circularity, prohibit the true intertwining of the newly paired strands. The formation of such nonintertwined or paranemic joints does not require superhelicity, and indeed can occur with relaxed closed circular DNA. E. coli topoisomerase I can intertwine the incoming single strand in the paranemic joint with its complement, thereby topologically linking single-stranded DNA to all of the duplex molecules in the reaction mixture. The efficiency of formation of paranemic joints, the time course, and estimates of their length, all suggest that they represent true synaptic intermediates in the pairing reaction promoted by RecA protein.

By searching processively RecA protein pairs DNA molecules that share a limited stretch of homology

RecA protein promotes homologous pairing by a reaction in which the protein first binds stoichiometrically to single-stranded DNA in a slow presynaptic step, and then conjoins single-stranded and duplex DNA, thereby forming a ternary complex. RecA protein did not pair molecules that shared only 30 bp homology, but, with full efficiency, it paired circular single-stranded and linear duplex molecules in which homology was limited to 151 bp at one end of the duplex DNA. The initial rate of the pairing reaction was directly related to the length of the heterologous part of the duplex DNA, which we varied from 0 to 3060 base pairs. Since interactions involving the heterologous part of a molecule speed the location of a small homologous region, we conclude that RecA protein promotes homologous alignment by a processive mechanism involving relative motion of conjoined molecules within the ternary complex.

Homologous pairing and topological linkage of DNA molecules by combined action of E. coli recA protein and topoisomerase I

E. coli RecA protein and topoisomerase I, acting on superhelical DNA and circular single strands in the presence of ATP and Mg2+, topologically link single-stranded molecules to one another, and single-stranded molecules to duplex DNA. When superhelical DNA is relaxed by prior incubation with topoisomerase, it is a poor substrate for catenation. Extensive homology stimulates the catenation of circular single-stranded DNA and superhelical DNA, whereas little reaction occurs between these forms of the closely related DNAs of phages phi X174 and G4, indicating that, in conjunction with topoisomerase I, RecA protein can discriminate perfect or nearly perfect homology from a high degree of relatedness. Circular single-stranded G4 DNA reacts with superhelical DNA of chimeric phage, M13G ori 1, to form catenanes, at least half of which survive heating at 80 degrees C following restriction cleavage in the M13 region, but few of which survive following restriction cleavage in the G4 region. Electron microscopic examination of catenated molecules cleaved in the M13 region reveals that in most cases the single-stranded G4 DNA is joined to the linear duplex M13(G4) DNA in the homologous G4 region. The junction frequently has the appearance of a D loop, with an extent equivalent to 100 or more bp. We conclude that a significant fraction of catenanes were hemicatenanes, in which the single-stranded circle was topologically linked, probably by multiple turns, to its complementary strand in the duplex DNA. These observations support the previous conclusion that RecA protein can pair a single strand with its complementary strand in duplex DNA in a side-by-side fashion without a free end in any of the three strands.

Rapid Exchange of A:T Base Pairs Is Essential for Recognition of DNA Homology by Human Rad51 Recombination Protein

Human Rad51 belongs to a ubiquitous family of proteins that enable a single strand to recognize homology in duplex DNA, and thereby to initiate genetic exchanges and DNA repair, but the mechanism of recognition remains unknown. Kinetic analysis by fluorescence resonance energy transfer combined with the study of base substitutions and base mismatches reveals that recognition of homology, helix destabilization, exchange of base pairs, and initiation of strand exchange are integral parts of a rapid, concerted mechanism in which A:T base pairs play a critical role. Exchange of base pairs is essential for recognition of homology, and physical evidence indicates that such an exchange occurs early enough to mediate recognition.