T4 RNA ligase has been shown to synthesize nucleoside and dinucleoside 5 H -polyphosphates by displacement of the AMP from the E-AMP complex with polyphosphates and nucleoside diphosphates and triphosphates. Displacement of the AMP by tripolyphosphate (P 3 ) was concentration dependent, as measured by SDS/PAGE. When the enzyme was incubated in the presence of 0.02 mm [a-32 P] ATP, synthesis of labeled Ap 4 A was observed: ATP was acting as both donor (K m , mm) and acceptor (K m , mm) of AMP from the enzyme. Whereas, as previously known, ATP or dATP (but not other nucleotides) were able to form the E-AMP complex, the specificity of a compound to be acceptor of AMP from the E-AMP complex was very broad, and with K m values between 1 and 2 mm. In the presence of a low concentration (0.02 mm) of [a-32 P] ATP (enough to form the E-AMP complex, but only marginally enough to form Ap 4 A) and 4 mm of the indicated nucleotides or P 3 , the relative rate of synthesis of the following radioactive (di)nucleotides was observed: Ap 4 X (from XTP, 100); Ap 4 dG (from dGTP, 74); Ap 4 G (from GTP, 49); Ap 4 dC (from dCTP, 23); Ap 4 C (from CTP, 9); Ap 3 A (from ADP, 5); Ap 4 ddA, (from ddATP, 1); p 4 A (from P 3 , 200). The enzyme also synthesized efficiently Ap 3 A in the presence of 1 mm ATP and 2 mm ADP. The following T4 RNA ligase donors were inhibitors of the synthesis of Ap 4 G: pCp . pAp . pA2 H p.

T4 DNA iigase (EC 6.5.1.1), one of the most widely used enzymes in genetic engineering, transfers AMP from the E-AMP complex to tripolyphosphate, ADP, ATP, GTP or dATP producing p4A, Ap3A, Ap4A, Ap4G and Ap4dA, respectively. Nicked DNA competes very effectively with GTP for the synthesis of ApIG and, conversely, tripolyphosphate (or GTP) inhibits the iigation of DNA by the ligase. As T4 DNA ligase has similar requirements for ATP as the mammalian DNA ligase(s), the latter enzyme(s) could also synthesize dinucleoside polyphosphates. The present report may be related to the recent finding that human Fhit (fragile histidine triad) protein, encoded by the FHIT putative tumor suppressor gene, is a typical dinucleoside 5',5"-Pa,pa-triphosphate (Ap3A) hydrolase (EC 3.6.1.29).

Background: In vivo treatment with growth hormone reduces radiation-associated mortality. The molecular mechanisms underlying this effect are unknown. It has been described that increased sensitivity to ionising radiation can be due to defects in machinery involved in detection and/or repair of DNA double-strand breaks. Objective: To study the mechanisms involved in growth hormone action on the increased survival in irradiated cells. Materials and methods: CHO-4 cells stably expressing the growth hormone receptor were used. A cell viability assay was carried out to analyse the increase in survival induced by growth hormone in irradiated cells. To investigate whether the DNA repair mechanism could be implicated in this effect we performed DNA reactivation assays using pHIV-LUC and pCMV-bgal plasmids as control. Identical studies were also conducted using the radiomimetic drug, bleomycin. Results: Growth hormone protects CHO-4 cells from bleomycin-and radiation-induced cell death. In pHIV-LUC transfected cells, a time-dependent decrease in luciferase activity was observed after irradiation in the absence of growth hormone. However, cells pretreated with this hormone maintained reporter activity. When cells were transfected with irradiated pHIV-LUC plasmid, only the hormone-treated cells recovered the transcriptional activity. Conclusions: Growth hormone exerts a radioprotective effect in CHO-4 cells stably transfected with the complementary DNA for the rat growth hormone receptor. The radioprotection is triggered directly by the hormone and it is also observed with bleomycin. The increased survival in response to radiation and bleomycin treatment induced by growth hormone correlates with an enhanced ability of the cells to repair damaged DNA.