Binding mode of porphyrins to poly[d(A-T)(2)] and poly[d(G-C)(2)

We examined the binding geometry of Co-meso-tetrakis (N-methyl pyridinium-4-yl)porphyrin, Co-meso-tetrakis (N-n-butyl pyridinium-4-yl)porphyrin and their metal-free ligands to poly[d(A-T)(2)] and poly[d(G-C)(2)] by optical spectroscopic methods including absorption, circular and linear dichroism spectroscopy, and fluorescence energy transfer technique. Signs of an induced CD spectrum in the Soret band depend only on the nature of the DNA sequence; all porphyrins exhibit negative CD when bound to poly[d(G-C)(2)] and positive when bound to poly[d(A-T)(2)]. Close analysis of the linear dichroism result reveals that all porphyrins exhibit outside binding when complexed with poly[d(A-T)(2)], regardless of the existence of a central metal and side chain. However, in the case of poly[d(G-C)(2)], we observed intercalative binding mode for two nonmetalloporphyrins and an outside binding mode for metalloporphyrins. The nature of the outside binding modes of the porphyrins, when complexed with poly[d(A-T)(2)] and poly[d(G-C)(2)], are quite different. We also demonstrate that an energy transfer from the excited nucleo-bases to porphyrins can occur for metalloporphyrins.     

Stopped-flow kinetic analysis of the interaction of anthraquinone anticancer drugs with calf thymus DNA, poly[d(G-C)].poly[d(G-C)], and poly[d(A-T)].poly[d(A-T)]

The sodium dodecyl sulfate driven dissociation reactions of daunorubicin (1), mitoxantrone (2), ametantrone (3), and a related anthraquinone without hydroxyl groups on the ring or side chain (4) from calf thymus DNA, poly[d(G-C)]2, and poly[d(A-T)]2 have been investigated by stopped-flow kinetic methods. All four compounds exhibit biphasic dissociation reactions from their DNA complexes. Daunorubicin and mitoxantrone have similar dissociation rate constants that are lower than those for ametantrone and 4. The effect of temperature and ionic strength on both rate constants for each compound is similar. An analysis of the effects of salt on the two rate constants for daunorubicin and mitoxantrone suggests that both of these compounds bind to DNA through a mechanism that involves formation of an initial outside complex followed by intercalation. The daunorubicin dissociation results from both poly[d(G-C)]2 and poly[d(A-T)]2 can be fitted with a single exponential function, and the rate constants are quite close. The ametantrone and 4 polymer dissociation results can also be fitted with single exponential curves, but with these compounds the dissociation rate constants for the poly[d(G-C)]2 complexes are approximately 10 times lower than for the poly[d(A-T)]2 complexes. Mitoxantrone also has a much slower dissociation rate from poly[d(G-C)]2 than from poly[d(A-T)]2, but its dissociation from both polymers exhibits biphasic kinetics. Possible reasons for the biphasic behavior with the polymers, which is unique to mitoxantrone, are selective binding and dissociation from the alternating polymer intercalation sites and/or dual binding modes of the intercalator with both side chains in the same groove or with one side chain in each groove.     

Right- and left-handed helixes of poly[d(A-T)].poly[d(A-T)] investigated by infrared spectroscopy

The secondary structures of double-stranded poly[d(A-T)].poly[d(A-T)] in films have been studied by IR spectroscopy with three different counterions (Na+, Cs+, and Ni2+) and a wide variety of water content conditions (relative humidity between 100 and 47%). In addition to the A-, B-, C-, and D-form spectra, a new IR spectrum has been obtained in the presence of nickel ions. The IR spectra of Ni2+-poly[d(A-T)].poly[d(A-T)] films are analyzed by comparison with previously assigned IR spectra of left-handed poly[d(G-C)].poly[d(G-C)] and poly[d(A-C)].poly[d(G-T)], and it is possible to conclude that they reflect a Z-type structure for poly[d(A-T)].poly[d(A-T)]. The Z conformation has been favored by the high polynucleotide concentration, by the low water content of the films, and by specific interactions of the transition metal ions with the purine bases stabilized in a syn conformation. A structuration of the water hydration molecules around the double-stranded Ni2+-poly[d(A-T)].poly[d(A-T)] is shown by the presence of a strong sharp water band at 1615 cm-1.     

DNA binding of a spermine derivative: Spectroscopic study of anthracene-9-carbonyl-n1-spermine with poly[d(G-C)·(d(G-C))] and poly[d(A-T) · d(A-T)]

The binding of polyamines, including spermidine ( 1 ) and spermine ( 2 ), to poly[d(G-C) · d(G-C) ] was probed using spectroscopic studies of anthracene-9-carbonyl-N¹-spermine ( 3 ); data from normal absorption, linear dichroism (LD), and circular dichroism (CD) are reported. Ligand LD and CD for transitions located in the DNA region of the spectrum were used. The data show that 3 binds to DNA in a manner characteristic of both its amine and polycyclic aromatic parts. With poly [(dG-dC) · (dG-dC)], binding modes are occupied sequentially and different modes correspond to different structural perturbations of the DNA. The most stable binding mode for 3 with poly[d(G-C) · d(G-C)] has a site size of 6 ± 1 bases, and an equilibrium binding constant of (2.2 ± 1.1) × 10⁷ M^?1 with the anthracene moiety intercalated. It dominates the spectra from mixing ratios of approximately 133:1 until 6:1 DNA phosphate: 3 is reached. The analogous data for poly [d(A-T) · d(A-T)] between mixing ratios 36:1 and 7:1 indicates a site size of 8.3 ± 1.1 bases and an equilibrium binding constant of (6.6 ± 3.3) × 10⁵ M^?1. Thus, 3 binds preferentially to poly [d(G-C) · d(G-C)] at these concentrations. © 1994 John Wiley & Sons, Inc.     

Raman spectroscopy of Z-form poly[d(A-T)].poly[d(A-T)

Helical structures of double-stranded poly[d(A-T)] in solution have been studied by Raman spectroscopy. While the classical right-handed conformation B-type spectra are obtained in the case of sodium chloride solutions, a Z-form Raman spectrum is observed by addition of nickel ions at high sodium concentration, conditions in which the inversion of the circular dichroic spectrum of poly[d(A-T)] is detected, similar to that observed for high-salt poly[d(G-C)] solutions [Bourtayre, P., Liquier, J., Pizzorni, L., & Taillandier, E. (1987) J. Biomol. Struct. Dyn. 5, 97-104]. The characterization of the Z-form spectrum of poly[d(A-T)] is proposed by comparison with previously obtained characteristic Raman lines of Z-form poly[d(G-C)] and poly[d(A-C)].poly[d(G-T)] solutions and of d(CG)3 and d(CGCATGCG) crystals [Thamann, T. J., Lord, R. C., Wang, A. H.-J., & Rich, A. (1981) Nucleic Acids Res. 9, 5443-5457; Benevides, J. M., Wang, A. H.-J., van der Marel, G. A., van Boom, J. H., Rich, A., & Thomas, G. J., Jr. (1984) Nucleic Acids Res. 14, 5913-5925]. Detailed spectroscopic data are presented reflecting the reorientation of the purine-deoxyribose entities (C2'-endo/anti----C3'-endo/syn), the modification of the phosphodiester chain, and the adenosine lines in the 1300-cm-1 region. The role played by the hydrated nickel ions in the B----Z transition is discussed.     

CD of the synthetic RNA duplexes poly[r(A-T)] and poly[r(A-U)] in salt and ethanolic solutions.

Synthetic RNA poly[r(A-T)] has been synthesized and its CD spectral properties compared to those of poly[r(A-U)], poly[d(A-T)], and poly[d(A-U)] in various salt and ethanolic solutions. The CD spectra of poly[r(A-T)] in an aqueous buffer and of poly[d(A-T)] in 70.8% v/v ethanol are very similar, suggesting that they both adopt the same A conformation. On the other hand, the CD spectra of poly[r(A-T)] and of poly[r(A-U)] differ in aqueous, and even more so in ethanolic, solutions. We have recently observed a two-state salt-induced isomerization of poly[r(A-U)] into chiral condensates, perhaps of Z-RNA [M. Vorlícková, J. Kypr, and T. M. Jovin, (1988) Biopolymers 27, 351-354]. It is shown here that poly[r(A-T)] does not undergo this isomerization. Both the changes in secondary structure and tendency to aggregation are different for poly[r(A-T)] and poly[r(A-U)] in aqueous salt solutions. In most cases, the CD spectrum of poly[r(A-U)] shows little modification of its CD spectrum unless the polymer denatures or aggregates, whereas poly[r(A-T)] displays noncooperative alterations in its CD spectrum and a reduced tendency to aggregation. At high NaCl concentrations, poly[r(A-T)] and poly[r(A-U)] condense into psi(-) and psi(+) structures, respectively, indicating that the type of aggregation is dictated by the polynucleotide chemical structure and the corresponding differences in conformational properties.     

The B-Z conformational transition and aggregation of poly[d(G-C)] induced by moderate concentrations of Mg(CIO4)2

Mg(ClO4)2 induces the cooperative B-to-Z transition of poly[d(G-C)]; the salt concentration at the midpoint is 0.26 M. A comparison with previous data for NaCl, MgCl2 and NaClO4 (F.M. Pohl and T.M. Jovin, J. Mol. Biol. 67 (1972) 375) indicates that Mg(ClO4)2 is more effective than would be anticipated from the simple additive effects of the Mg2+ and ClO4- ions (the ionic strengths of the respective transition points are: NaCl, 2.4; MgCl2, 2.1; NaClO4, 1.8 and Mg(ClO4)2, 0.78). These results suggest the importance of specific interactions involving ClO4-, particularly in the presence of Mg2+. The B-Z transition of poly[d(G-C)] can be monitored spectroscopically via the large hyperchromic shift at 295 nm and the inversion in the CD spectrum. The reaction is fully reversible and can be fitted by a monoexponential function with half times varying between 8 and 150 min. The observed relaxation times are strongly dependent on the concentration of Mg(ClO4)2 with a distinct maximum at the transition point, in accordance with a concerted mechanism involving only the B and Z states. As the polymer assumes the Z conformation it progressively aggregates into a gel-like precipitate, which, however, redissolves rapidly upon lowering the salt concentration. The natural DNA from Micrococcus lysodeikticus which has a high GC content of 72% is also precipitated by Mg(ClO4)2 but we do not have direct spectroscopic evidence for the involvement of the Z conformation in this phenomenon. Neither calf thymus DNA (41% GC) nor poly[d(A-T)] (0% GC) aggregates under the same conditions.     

fd gene 5 protein binds to double-stranded polydeoxyribonucleotides poly(dA.dT) and poly[d(A-T).d(A-T)]

Circular dichroism (CD) data indicated that fd gene 5 protein (G5P) formed complexes with double-stranded poly(dA.dT) and poly[d(A-T).d(A-T)]. CD spectra of both polymers at wavelengths above 255 nm were altered upon protein binding. These spectral changes differed from those caused by strand separation. In addition, the tyrosyl 228-nm CD band of G5P decreased more than 65% upon binding of the protein to these double-stranded polymers. This reduction was significantly greater than that observed for binding to single-stranded poly(dA), poly(dT), and poly[d(A-T)] but was similar to that observed for binding of the protein to double-stranded RNA [Gray, C.W., Page, G.A., & Gray, D.M. (1984) J. Mol. Biol. 175, 553-559]. The decrease in melting temperature caused by the protein was twice as great for poly[d(A-T).d(A-T)] as for poly(dA.dT) in 5 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH 7. Upon heat denaturation of the poly(dA.dT)-G5P complex, CD spectra showed that single-stranded poly(dA) and poly(dT) formed complexes with the protein. The binding of gene 5 protein lowered the melting temperature of poly(dA.dT) by 10 degrees C in 5 mM Tris-HCl, pH 7, but after reducing the binding to the double-stranded form of the polymer by the addition of 0.1 M Na+, the melting temperature was lowered by approximately 30 degrees C. Since increasing the salt concentration decreases the affinity of G5P for the poly(dA) and poly(dT) single strands and increases the stability of the double-stranded polymer, the ability of the gene 5 protein to destabilize poly(dA.dT) appeared to be significantly affected by its binding to the double-stranded form of the polymer.     

Z form of poly d(A-T).poly d(A-T) in solution studied by CD and UV spectroscopies

Near UV CD spectra, UV absorption spectra and their first derivatives have been recorded on poly d(A-T).poly d(A-T) solutions in presence of high NaCl concentration and various amounts of NiCl2. Comparison of the results presented here with those obtained for poly d(G-C).poly d(G-C) and poly d(A-C).poly d(G-T) in comparable conditions, and the I.R. and Raman data on poly d(A-T).poly d(A-T), allows us to assign the new spectra to the Z conformation of poly d(A-T).poly d(A-T) in solution. The mechanism by which nickel ions induce the B----Z interconversion in the presence of high NaCl concentration is discussed.     

Interaction of lac repressor with alternating poly d (A-T) and poly d (G-C). Circular dichroism studies

The binding of lac repressor to poly d(A-T) and poly d(G-C) has been studied using circular dichroism. The results indicate that the binding induces the same conformational change of both polynucleotides and perturbs the same number of nucleic acid bases (28 bases). It is shown that in 0.1 M phosphate buffer the CD measurement can be used to determine the binding constant of lac repressor to poly d(A-T). Competition experiments performed at various salt concentrations show that the stronger interaction of lac repressor for poly d(A-T) than for poly d(G-C) is based on difference in the dissociation rate of the complexes whereas the association rate for both polymers are similar.     

Conformational properties of poly[d(G-T)].poly[d(C-A)] and poly[d(A-T)] in low- and high-salt solutions: NMR and laser Raman analysis

³¹P- and ¹H-nmr and laser Raman spectra have been obtained for poly[d(G-T)]·[d(C-A)] and poly[d(A-T)] as a function of both temperature and salt. The ³¹P spectrum of poly[d(G-T)]·[d(C-A)] appears as a quadruplet whose resonances undergo separation upon addition of CsCl to 5.5M. ¹H-nmr measurements are assigned and reported as a function of temperature and CsCl concentration. One dimensional nuclear Overhauser effect (NOE) difference spectra are also reported for poly[d(G-T)]·[d(C-A)] at low salt. NOE enhancements between the H8 protons of the purines and the C5 protons of the pyrimidines, (H and CH₃) and between the base and H-2′,2″ protons indicate a right-handed B-DNA conformation for this polymer. The NOE patterns for the TH3 and GH1 protons in H₂O indicate a Watson–Crick hydrogen-bonding scheme. At high CsCl concentrations there are upfield shifts for selected sugar protons and the AH2 proton. In addition, laser Raman spectra for poly[d(A-T)] and poly[d(G-T)]·[d(C-A)] indicate B-type conformations in low and high CsCl, with predominantly C2′-endo sugar conformations for both polymers. Also, changes in base-ring vibrations indicate that Cs⁺ binds to O2 of thymine and possibly N3 of adenine in poly[d(G-T)]·[d(C-A)] but not in poly[d(A-T)]. Further, ¹H measurements are reported for poly[d(A-T)] as a function of temperature in high CsCl concentrations. On going to high CsCl there are selective upfield shifts, with the most dramatic being observed for TH1′. At high temperature some of the protons undergo severe changes in linewidths. Those protons that undergo the largest upfield shifts also undergo the most dramatic changes in linewidths. In particular TH1′, TCH₃, AH1′, AH2, and TH6 all undergo large changes in linewidths, whereas AH8 and all the H-2′,2″ protons remain essentially constant. The maximum linewidth occurs at the same temperature for all protons (65°C). This transition does not occur for d(G-T)·d(C-A) at 65°C or at any other temperature studied. These changes are cooperative in nature and can be rationalized as a temperature-induced equilibrium between bound and unbound Cs⁺, with duplex and single-stranded DNA. NOE measurements for poly[d(A-T)] indicate that at high Cs⁺ the polymer is in a right-handed B-conformation. Assignments and NOE effects for the low-salt ¹H spectra of poly[d(A-T)] agree with those of Assa-Munt and Kearns [(1984) Biochemistry 23 , 791–796] and provide a basis for analysis of the high Cs⁺ spectra. These results indicate that both polymers adopt a B-type conformation in both low and high salt. However, a significant variation is the ability of the phosphate backbone to adopt a repeat dependent upon the base sequence. This feature is common to poly[d(G-T)]·[d(C-A)], poly[d(A-T)], and some other pyr–pur polymers [J. S. Cohen, J. B. Wouten & C. L Chatterjee (1981) Biochemistry 20 , 3049–3055] but not poly[d(G-C)].     

Poly(dA).poly(dT) exists in an unusual conformation under physiological conditions: propidium binding to poly(dA).poly(dT) and poly[d(A-T)].poly[d(A-T)]

The binding of propidium to poly(dA).poly(dT) [poly(dA.dT)] and to poly[d(A-T)].poly[d(A-T)] [poly[d(A-T)2]] has been compared under a variety of solution conditions by viscometric titrations, binding studies, and kinetic experiments. The binding of propidium to poly[d(A-T)2] is quite similar to its binding to calf thymus deoxyribonucleic acid (DNA). The interaction with poly(dA.dT), however, is quite unusual. The viscosity of a poly(dA.dT) solution first decreases and then increases in a titration with propidium at 18 degrees C. The viscosity of poly[d(A-T)2] shows no decrease in a similar titration. Scatchard plots for the interaction of propidium with poly(dA.dT) show the classical upward curvature for positive cooperativity. The curvature decreases as the temperature is increased in binding experiments. A van't Hoff plot of the observed binding constants yields an apparent positive enthalpy of approximately +6 kcal/mol for the propidium-poly(dA.dT) interaction. Propidium binding to poly[d(A-T)2] shows no evidence for positive cooperativity, and the enthalpy change for the reaction is approximately -9 kcal/mol. Both the magnitude of the dissociation constants and the effects of ionic strength are quite similar for the dissociation of propidium from poly(dA-T)2] and from poly[d(A-T)2], suggesting that the intercalated states are similar for the two complexes. The observed association reactions, under pseudo-first-order conditions, are quite different. Plots of the observed pseudo-first-order association rate constant vs. polymer concentration have much larger slopes for propidium binding to poly[d(A-T)2] than to poly(dA.dT).(ABSTRACT TRUNCATED AT 250 WORDS)     

Anomalous excitonic CD of meso-tetrakis(3-N-methylpyridiniumyl)porphyrin bound to poly[d(A-T)(2)

When meso-tetrakis(3-N-methylpyridiniumyl)porphyrin (m-TMPyP) formed a complex with poly[d(A-T)(2)], an intense bisignate excitonic CD in the Soret absorption region was observed. The excitonic CD of the m-TMPyP-poly[d(A-T)(2)] complex is unique in that no other combination of the related porphyrin, namely, meso-tetrakis(n-N-methylpyridiniumyl)porphyrin (where n = 2, 4), and polynucleotide including calf thymus DNA, poly[d(G-C)(2)], poly[d(I-C)(2)], and poly(dA).poly(dT), exhibits a comparable CD spectrum. From the [drug]/[DNA] ratio-dependence of the intensity and the shape of the CD spectrum, this porphyrin species is assigned to an extensively aggregated form. The extensively aggregated porphyrin disperses in 1 h after mixing to form moderately stacked porphyrin at a low mixing ratio. The magnitude of linear dichroism of the extensively aggregated porphyrin was small and the sign was negative in the Soret band, which indicated that the molecular plane of porphyrin in the complex is strongly tilted. On the other hand, the molecular plane of porphyrin is almost parallel to the DNA base plane (perpendicular to the DNA helix axis) in the moderately stacked form.     

Energetics of Z-DNA formation in poly d(A-T), poly d(G-C), and poly d(A-C) poly d(G-T).

The conformational change for the alternating purine-pyrimidine polydeoxyribonucleotides i.e. poly d(A-T), poly d(G-C), and poly d(A-C) poly d(G-T) from a right-handed conformation at room temperature to the left-handed Z-DNA like double helix at elevated temperatures has been studied by UV spectroscopy, Raman spectroscopy, and by adiabatic differential scanning microcalorimetry (DSC) in the presence of Na+ and Mg2+ or Ni2+ respectively as counterions. The differential UV spectra reveal through a hyperchromic shift at around 280nm and a hypochromic shift at 260nm that a conformational change to the left-handed conformation occurs. The Raman spectra clearly show characteristic changes, a drastic decrease of the band at 680cm-1 and the appearance of a new band at 628cm-1, due to the change of the purine bases to the syn conformation upon inversion of the helix-handedness. The course of the transition as function of temperature can be followed quantitatively by plotting the change in the excess heat capacity vs. temperature. The transition enthalpy delta H for the B- to Z-DNA transition per mole base pairs (mbp) amounts to 2.0 +/- 0.2kcal for poly d(G-C), to 4.0 +/- 0.4kcal for poly d(A-T), and to 3.1 +/- 0.3kcal for poly d(A-C) poly d(G-T). The enthalpy change due to the Z-DNA to coil transitions (per mole base pairs) amounts to 11kcal for poly d(G-C), 10.5kcal for poly d(A-T) and 11.3kcal for poly d(A-C) poly d(G-T).     

Circular dichroism of two conformations of poly[d(G-C)] induced by low pH.

Circular dichroism (CD) and UV absorption data showed that poly[d(G-C)] (at 0.09M NaCl, 0.01M Na+ (phosphate), 20 degrees C) underwent two conformational transitions upon lowering of the pH by the addition of HCl. The first transition was complete at about pH 3.0. The second transition was complete upon lowering the pH to 2.6 or upon raising the temperature, at pH 3.0, to about 40 degrees C. There was no indication of denaturation during either transition. The CD spectrum for the second acid conformation had large CD bands including a positive one at 288nm, a characteristic associated with C X C+ base-pairs. Electron microscopy showed no significant formation of condensed supramolecular aggregates corresponding to the first or second acid forms of poly[d(G-C)]. On the basis of spectral data, electron microscopy, and proton-uptake measurements, we propose models for the secondary structures that poly[d(G-C)] adopts in its two acid conformations.     

Temperature dependence of the volumetric parameters of drug binding to poly[d(A-T)].Poly[d(A-T)] and Poly(dA).Poly(dT)

We report the temperature and salt dependence of the volume change (DeltaVb) associated with the binding of ethidium bromide and netropsin with poly(dA).poly(dT) and poly[d(A-T)].poly[d(A-T)]. The DeltaV(b) of binding of ethidium with poly(dA).poly(dT) was much more negative at temperatures approximately 70 degrees C than at 25 degrees C, whereas the difference is much smaller in the case of binding with poly[d(A-T)].poly[d(A-T)]. We also determined the volume change of DNA-drug interaction by comparing the volume change of melting of DNA duplex and DNA-drug complex. The DNA-drug complexes display helix-coil transition temperatures (Tm several degrees above those of the unbound polymers, e.g., the Tm of the netropsin complex with poly(dA)poly(dT) is 106 degrees C. The results for the binding of ethidium with poly[d(A-T)].poly[d(A-T)] were accurately described by scaled particle theory. However, this analysis did not yield results consistent with our data for ethidium binding with poly(dA).poly(dT). We hypothesize that heat-induced changes in conformation and hydration of this polymer are responsible for this behavior. The volumetric properties of poly(dA).poly(dT) become similar to those of poly[d(A-T)].poly[d(A-T)] at higher temperatures.     

Factor C from rabbit liver. A new poly(dC) and poly[d(G-C)] template-selective stimulatory protein of DNA polymerases

We have undertaken a search for mammalian DNA-binding proteins that enhance the activity of DNA polymerases in a template sequence-specific fashion. In this paper, we report the extensive purification and characterization of a new DNA-binding protein from rabbit liver that selectively stimulates DNA polymerases to copy synthetic poly[d(G-C)] and the poly(dC) strand of poly(dC).poly(dG) as well as single-stranded natural DNA that contains stretches of oligo(dC). The enhancing protein, a polypeptide of 65 kDa designated factor C, stimulates the copying of the two synthetic templates by Escherichia coli DNA polymerase I, Micrococcus luteus polymerase, and eukaryotic DNA polymerases alpha and beta, but not by avian myeloblastosis virus polymerase. Factor C, however, does not affect utilization by these polymerases of the poly(dG) strand of poly(dC).poly(dG), of poly(dC) primed by oligo(dG), or of poly(dA).poly(dT) and poly[d(A-T)]. With polymerase I, Michaelis constants (Km) of poly[d(G-C)] and of the poly(dC) strand of poly(dC).poly(dG) are decreased by factor C 37- and 4.7-fold, respectively, whereas maximum velocity (Vmax) remains unchanged. By contrast, neither the Km value of the poly(dG) strand of poly(dC).poly(dG) nor the Vmax value with this template is altered by factor C. Rates of copying of activated DNA, denatured DNA, or singly primed M13 DNA are not affected significantly by factor C. However, primer extension analysis of the copying of recombinant M13N4 DNA that contains runs of oligo(dC) within an inserted thymidine kinase gene shows that factor C increases processivity by specifically augmenting the efficiency at which polymerase I traverses the oligo(dC) stretches. Direct binding of factor C to denatured DNA is indicated by retention of the protein-DNA complex on columns of DEAE-cellulose. Binding of factor C to poly[d(G-C)] is demonstrated by the specific adsorption of the enhancing protein to columns of poly[d(G-C)]-Sepharose. We propose that by binding to poly[d(G-C)] and to poly(dC).poly(dG), factor C enables tighter binding of some DNA polymerases to these templates and facilitates enzymatic activity.     

CD evidence that the alternating purine-pyrimidine sequence poly[d(A-C).d(G-T)], but not poly[d(A-T).d(A-T)], undergoes an acid-induced transition to a modified secondary conformation.

Circular dichroism and UV absorption data showed that poly[d(A-C).d(G-T)] (at 0.01M Na+ (phosphate), 20 degrees C) underwent two reversible conformational transitions upon lowering of the pH. The first transition was complete at about pH 3.9 and resulted in an acid form of the polymer that was most likely a modified, protonated duplex. The second transition occurred between pH 3.9 and 3.4 and consisted of the denaturation of this protonated duplex to the single strands. UV absorption and CD data also showed that the separated poly[d(A-C)] strand formed two acid-induced self-complexes with pKa values of 6.1 and 4.7 (at 0.01M Na+). However, neither one of these poly[d(A-C)] self-complexes was part of the acid-induced rearrangements of the duplex poly[d(A-C).d(G-T)]. Acid titration of the separated poly[d(G-T)] strand, under similar conditions, did not show the formation of any protonated poly[d(G-T)] self-complexes. In contrast to poly[d(A-C).d(G-T)], poly[d(A-T).d(A-T)] underwent only one acid-induced transition, which consisted of the denaturation of the duplex to the single strands, as the pH was lowered from 7 to 3.     

Circular dichroism studies of the interaction of a limited hydrolysate of T4 gene 32 protein with T4 DNA and poly[d(A-T)].poly[d(A-T)].

gp32 I is a protein with a molecular weight of 27 000. It is obtained by limited hydrolysis of T4 gene 32 coded protein, which is one of the DNA melting proteins. gp32 I itself appears to be also a melting protein. It denatures poly[d(A-T)].poly[d(A-T)] and T4 DNA at temperatures far (50-60 degrees C) below their regular melting temperatures. Under similar conditions gp32 I will denature poly[d(A-T).poly[d(A-T)] at temperatures approximately 12 degrees C lower than those measured for the intact gp32 denaturation. For T4 DNA gp32 shows no melting behavior while gp32 I shows considerable denaturation (i.e., hyperchromicity) even at 1 degree C. In this paper the denaturation of poly[d(A-T)].poly[d(A-T)] and T4 DNA by gp32 I is studied by means of circular dichroism. It appears that gp32 I forms a complex with poly[d(A-T)]. The conformation of the polynucleotide in the complex is equal to that of one strand of the double-stranded polymer in 6 M LiCl. In the gp32 I DNA complex formed upon denaturation of T4 DNA, the single-stranded DNA molecule has the same conformation as one strand of the double-strand T4 DNA molecule in the C-DNA conformation.