Terminal deoxynucleotidyl transferase catalyzes the reaction of DNA phosphorylation

Abstract

The reaction of phosphorylation and phosphonylation of an oligodeoxynucleotide 3'-terminal hydroxyl (oligodeoxynucleotidyl kinase activity) catalyzed by calf thymus terminal deoxynucleotidyl transferase (TDT) was found. Triphosphates modified at Palpha-, Palpha,gamma- or Palpha,beta,gamma-residues served as low-molecular weight substrates. The reaction was TDT specific; human DNA polymerasesalphaandbeta, as well as AMV reverse transcriptase did not catalyze it. The donor activity of modified triphosphates or triphosphonates depended on their structure and was increased with an increase in their hydrophobicity. The substrate activity of some modified triphosphates was up to one order of magnitude higher than that of ddTTP.

Figure 1

Structure of the tested compounds.

Figure 2

Primer extension by compounds IIIa–d catalyzed by TDT_P– (Series A, lanes 1–14) and TDT_P+ (Series A, lanes 15–20, Series B, lanes 1–4). (A) Lane 1, primer + enzyme (control); lane 2, as in lane 1 + 0.5 µM ddTTP; lanes 3–6, as in lane 1 + 2.5, 10, 25 and 100 µM IIIa, respectively; lanes 7–10, as in lane 1 + 2.5, 10, 25 and 100 µM IIIb, respectively; lanes 11–14, as in lane 1 + 0.025, 0.1, 0.25 and 1 µM IIId, respectively; lanes 15–17, as in lane 1 + 0.01, 0.1 and 1 µM IIId, respectively; lanes 18–20, as in lane 1 + 1, 10 and 100 µM IIIc, respectively. (B) Lane 1, primer + enzyme (control); lane 2, as in lane 1 + 0.2 µM ddTTP; lane 3, as in lane 1 + 5 µM IIId; lane 4, 3′-phosphorylated 14mer oligodeoxynucleotide VII.

Figure 2

Primer extension by compounds IIIa–d catalyzed by TDT_P– (Series A, lanes 1–14) and TDT_P+ (Series A, lanes 15–20, Series B, lanes 1–4). (A) Lane 1, primer + enzyme (control); lane 2, as in lane 1 + 0.5 µM ddTTP; lanes 3–6, as in lane 1 + 2.5, 10, 25 and 100 µM IIIa, respectively; lanes 7–10, as in lane 1 + 2.5, 10, 25 and 100 µM IIIb, respectively; lanes 11–14, as in lane 1 + 0.025, 0.1, 0.25 and 1 µM IIId, respectively; lanes 15–17, as in lane 1 + 0.01, 0.1 and 1 µM IIId, respectively; lanes 18–20, as in lane 1 + 1, 10 and 100 µM IIIc, respectively. (B) Lane 1, primer + enzyme (control); lane 2, as in lane 1 + 0.2 µM ddTTP; lane 3, as in lane 1 + 5 µM IIId; lane 4, 3′-phosphorylated 14mer oligodeoxynucleotide VII.

Figure 3

Primer extension by compounds III–IV. Lane 1, primer + TDT_P+ (control); lane 2, as in lane 1 + 10 µM dTTP; lanes 3–5, as in lane 2 + 50, 100 and 300 µM IIIb, respectively; lanes 6–8, as in lane 2 + 50, 100 and 300 µM IVa, respectively; lanes 9–11, as in lane 2 + 50, 100 and 300 µM IIIc, respectively; lanes 12–14, as in lane 2 + 50, 100 and 300 µM IVb, respectively.

Figure 4

Primer extension by compounds V catalyzed by TDT_P– (Series A) and TDT_P+ (Series B). (A) Lane 1, primer + enzyme (control); lanes 2–6, as in lane 1 + 0.0005, 0.001, 0.01, 0.1 and 1 µM Va, respectively; lanes 7–9, as in lane 1 + 0.001, 0.01 and 0.1 µM Vc, respectively; lanes 10–14, as in lane 1 + 0.001, 0.01, 0.1, 1 and 10 µM Vb, respectively. (B) Lane 1, primer + enzyme (control); lane 2, as in lane 1 + 5 µM dTTP; lanes 3–5, as in lane 2 + 5, 10 and 20 µM IIId, respectively; lanes 6–8, as in lane 2 + 0.5, 2 and 10 µM Va, respectively; lane 9, as in lane 1 + 2 µM ddTTP.

Figure 4

Primer extension by compounds V catalyzed by TDT_P– (Series A) and TDT_P+ (Series B). (A) Lane 1, primer + enzyme (control); lanes 2–6, as in lane 1 + 0.0005, 0.001, 0.01, 0.1 and 1 µM Va, respectively; lanes 7–9, as in lane 1 + 0.001, 0.01 and 0.1 µM Vc, respectively; lanes 10–14, as in lane 1 + 0.001, 0.01, 0.1, 1 and 10 µM Vb, respectively. (B) Lane 1, primer + enzyme (control); lane 2, as in lane 1 + 5 µM dTTP; lanes 3–5, as in lane 2 + 5, 10 and 20 µM IIId, respectively; lanes 6–8, as in lane 2 + 0.5, 2 and 10 µM Va, respectively; lane 9, as in lane 1 + 2 µM ddTTP.

Figure 5

Primer extension by compounds VI catalyzed by TDT_P+ (Series A) and TDT_P– (Series B). (A) Lane 1, primer+ enzyme (control); lane 2, as in lane 1 + 0.5 µM ddTTP; lane 3, as in lane 1 + 5 µM dTTP; lanes 4–6, as in lane 1 + 5, 50 and 500 µM VIa, respectively; lane 7, synthetic 3′-methylphosphonylated oligodeoxynucleotide VIII. (B) Lane 1, primer + enzyme (control); lane 2, as in lane 1 + 0.1 µM ddTTP; lane 3, as in lane 1 + 1 µM ddTTP and 10 µM dTTP; lanes 4–6, as in lane 1 + 100, 300 and 600 µM VIb, respectively; lanes 7 and 8, as in lane 6, primer extension reaction followed by addition of 0.05 and 0.1 M DTT, respectively; lanes 9–11, as in lane 1 + 100, 300 and 600 µM VIc, respectively; lanes 12 and 13, as in lane 11, primer extension reaction followed by addition of 0.05 and 0.1 M DTT, respectively; lanes 14–17, as in lane 1 + 100, 300, 600 and 1000 µM VId, respectively.

Figure 5

Primer extension by compounds VI catalyzed by TDT_P+ (Series A) and TDT_P– (Series B). (A) Lane 1, primer+ enzyme (control); lane 2, as in lane 1 + 0.5 µM ddTTP; lane 3, as in lane 1 + 5 µM dTTP; lanes 4–6, as in lane 1 + 5, 50 and 500 µM VIa, respectively; lane 7, synthetic 3′-methylphosphonylated oligodeoxynucleotide VIII. (B) Lane 1, primer + enzyme (control); lane 2, as in lane 1 + 0.1 µM ddTTP; lane 3, as in lane 1 + 1 µM ddTTP and 10 µM dTTP; lanes 4–6, as in lane 1 + 100, 300 and 600 µM VIb, respectively; lanes 7 and 8, as in lane 6, primer extension reaction followed by addition of 0.05 and 0.1 M DTT, respectively; lanes 9–11, as in lane 1 + 100, 300 and 600 µM VIc, respectively; lanes 12 and 13, as in lane 11, primer extension reaction followed by addition of 0.05 and 0.1 M DTT, respectively; lanes 14–17, as in lane 1 + 100, 300, 600 and 1000 µM VId, respectively.

Figure 6

Primer extension by dTTP, ddTTP, IIId and Va, catalyzed by TDT_P– (Series A, B) and TDT_P+ (Series C, D) in the presence of 2 mM Co²⁺ buffer (Series A, C) or 10 mM Mn²⁺ buffer (Series B, D). Lane 1, primer + enzyme (control); lanes 2–5, as in lane 1 + 5 µM dTTP, 0.1 µM ddTTP, 0.1 µM Va and 0.1 µM IIId, respectively.