Heteropolynucleotide Synthesis with Terminal Deoxyribonucleotidyltransferase<sup>*</sup>

Circular dichroism measurements were used to study the binding of fd gene 5 protein to fd DNA, to six polydeoxynucleotides (poly[d(A)], poly[d(T)], poly[d(I)], poly[d(C)], poly[d(A-T)], and the random copolymer poly[d(A,T)]), and to three oligodeoxynucleotides (d(pA)20, d(pA)7, and d(pT)7). Titrations of these DNAs with fd gene 5 protein were generally done in a low ionic strength buffer (5 mM Tris-HCl, pH 7.0 or 7.8) to insure tight binding, needed to obtain stoichiometric endpoints. By monitoring the CD of the nucleic acids above 250 nm, where the protein has no significant intrinsic optical activity, we found that there were two modes of binding, with the number of nucleotides covered by a gene 5 protein monomer (n) being close to either 4 or 3. These stoichiometries depended upon which polymer was titrated as well as upon the protein concentration. Single endpoints at nucleotide/protein molar ratios close to 3 were found during titrations of poly[d(T)] and fd DNA (giving n = 3.1 and 2.8 +/- 0.2, respectively), while CD changes with two apparent endpoints at nucleotide/protein molar ratios close to 4 and approximately 3 were found during titrations of poly[d(A)], poly[d(I)], poly[d(A-T)], and poly[d(A,T)] (with the first endpoints giving n = 4.1 4.0, 4.0, and 4.1 +/- 0.3, respectively). Calculations showed that the CD changes we observed during these latter titrations were consistent with a switch between two non-interacting binding modes of n = 4 and n = 3. We found no evidence for an n = 5 binding mode. One implication of our results is that the Brayer and McPherson model for the helical gene 5 protein-DNA complex, which has 5 nucleotides bound per protein monomer (G. Brayer and A. McPherson, J. Biomol. Struct. and Dyn. 2, 495-510, 1984), cannot be correct for the detailed solution structure of the complex. We interpreted the CD changes above 250 nm upon binding of the gene 5 protein to single-stranded DNAs to be the result of a slight unstacking of the bases, along with a significant alteration of the CD contributions of the individual nucleotides in the case of A-and/or T-containing DNAs. Interestingly, CD contributions attributed to nearest-neighbor interactions in free poly[d(A-T)], poly[d(A,T)], poly[d(A)], and poly[d(T)] were partially maintained in the CD spectra of the protein-saturated polymers, so that neighboring nucleotides, when bound to the protein at 20 degrees C, appeared to interact with one another in much the same manner as in the free polymers at 50 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…Fd DNA was either isolated as in previous work (8) (23,24) . The poly[d(l)] was a portion of a sample used in previous work (25).…”

Section: Nucleic Acidsmentioning

confidence: 99%

“…The poly[d(l)] was a portion of a sample used in previous work (25). The poly[d(A,T)] was newly synthesized using the procedure of Ratliff et al (24) and , by incorporation of radioactive tracers, was shown to contain 51 % thymidylate and 49% adenylate. (26).…”

Section: Nucleic Acidsmentioning

confidence: 99%

The Binding of fd Gene 5 Protein to Polydeoxynucleotides: Evidence from CD Measurements for Two Binding Modes

Kansy

Clack

Gray

1986

Journal of Biomolecular Structure and Dynamics

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“…These heating curves are exactly reversible on cooling, and they can be recycled more than once with no perceptible change. The corrected melting temperatures as determined from the peaks of the calculated differential curves [20] are 19.5" for the dAAC-dT pair and 52.5" for the dA-dT pair. …”

Section: Mixtures With D(t)6(aac)z (Daac-dt) and With D(t)(a)m (Da-dt)mentioning

confidence: 99%

“…However, when dG units were enzymatically copolymerized with any other nucleotide or combination of nucleotides, aggregation was absent [20]. Aggregation of dC units interferes with use of dC oligomers longer than decamer as initiators for chain lengthening [21].…”

mentioning

confidence: 99%

Synthesis of N‐Acetylated Deoxyribonucleoside 5′‐Triphosphates and their Utilization in Enzymatic Formation of Single‐Stranded Polydeoxyribonucleotides

Hayes¹,

Hansbury²,

Mitchell³

et al. 1968

European Journal of Biochemistry

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The N-acetylated deoxyadenosine and deoxyguanosine 5'4riphosphates (dAAcTP and dGAcTP) have been synthesized and found competent as substrates for enzymatic chain lengthening of polydeoxyribonucleotides without concomitant loss of the acetyl group. The enzyme used was terminal deoxyribonucleotidyltransferase. The addition of dGAc units proceeds without aggregation that self-limits addition of nonacetylated dG units. The resulting polymer 3'-ended with dGAC units is an efficient initiator for subsequent chain lengthening. Hydrogen bonding is not destroyed by N-acetylation of the dA-dT Watson-Crick pair, although it is considerably weakened. As a practical result of this, a polymer 3'-ended with dAAC units is a much more efficient initiator for enzymatic chain lengthening by dT units than is a corresponding polymer with dA units. Venom phosphodiesterase does not hydrolyze a polymer 3'-ended with dAAc U n i t s .Synthesis of polydeoxyribonucleotides with desired sequences can be a key contributor to successful, designed cellular transformation. According to current knowledge about the chemistry of the cell genome, the arrangement of nucleotide units is a linear mixture of hetero-and homopolymer sequences (CBN) ; overlining of a subscript means that the number is the average of a distribution; the superscripts Ac or Ac, following a capital letter show that the nucleotide is acetylated or diacetylated; monomer units in a polymer are referred to by dA, dG*c, etc.; labeling in compounds is sparingly designated, usually just a t the start of an experiment ; the mono-ymole is a micromole of monomer units contained in a polymer. 33.tion of dG units onto an initiator but also very inefficient utilization as an initiator of an oligodeoxyribonucleotide 3'-terminated with a run of dG units. However, when dG units were enzymatically copolymerized with any other nucleotide or combination of nucleotides, aggregation was absent [20]. Aggregation of dC units interferes with use of dC oligomers longer than decamer as initiators for chain lengthening [21]. Hydrogen bonding slows the rate of dT unit addition onto poly dA initiators [22].It has been shown that N-acetylated dG oligomers do not have the property of aggregation [17], and it may readily be inferred that this same chemical modification on other amino-bearing nucleotides could eliminate the other difficulties mentioned above. Therefore, we have synthesized the N-acetylated 5'-triphosphates dGAcTP and dAACTP in order to determine whether these can function as substrates for enzymatic chain lengthening, whether the products retain the N-acetyl group and are devoid of the aggregation and hydrogen-bonding difficulties, and whether the N-acetyl group is Gnally removable with no damage to the polydeoxyribonucleotide. METHODS AND N A T E R

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“…Polymerization of mixtures of deoxynucleoside triphosphates by termi nal deoxynucleotidyltransferase of calf thymus yields polymers without rec ognizable order in sequence (121)(122)(123)(124). The sequence is, however, influ enced by the identity of the nucleotide substrates as well as the stoichiome try of the mixture.…”

mentioning

confidence: 99%

Biosynthesis of DNA

Goulian¹

1971

Annu. Rev. Biochem.

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Heteropolynucleotide Synthesis with Terminal Deoxyribonucleotidyltransferase^*

Cited by 20 publications

References 16 publications

The Binding of fd Gene 5 Protein to Polydeoxynucleotides: Evidence from CD Measurements for Two Binding Modes

The Binding of fd Gene 5 Protein to Polydeoxynucleotides: Evidence from CD Measurements for Two Binding Modes

Synthesis of N‐Acetylated Deoxyribonucleoside 5′‐Triphosphates and their Utilization in Enzymatic Formation of Single‐Stranded Polydeoxyribonucleotides

Biosynthesis of DNA

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Heteropolynucleotide Synthesis with Terminal Deoxyribonucleotidyltransferase*

Cited by 20 publications

References 16 publications

The Binding of fd Gene 5 Protein to Polydeoxynucleotides: Evidence from CD Measurements for Two Binding Modes

The Binding of fd Gene 5 Protein to Polydeoxynucleotides: Evidence from CD Measurements for Two Binding Modes

Synthesis of N‐Acetylated Deoxyribonucleoside 5′‐Triphosphates and their Utilization in Enzymatic Formation of Single‐Stranded Polydeoxyribonucleotides

Biosynthesis of DNA

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Heteropolynucleotide Synthesis with Terminal Deoxyribonucleotidyltransferase^*