2 ATP = diadenosine 5',5''''-P1,P4-tetraphosphate + diphosphate
overall reaction. LysU possesses a dual catalytic activity, initially producing 5’,5’’’-P1,P4-tetraphosphate from ATP, before converting that tetraphosphate to a triphosphate (diadenosine 5’,5’’’-P1,P3-triphosphate (Ap3A) synthase activity). It is shown that 5’,5’’’-P1,P3-triphosphate formation requires (1) that the second step of 5’,5’’’-P1,P4-tetraphosphate formation is slightly reversible, leading to a reappearance of adenylate intermediate, and (2) that phosphate is present to trap the intermediate. 5’,5’’’-P1,P3-triphosphate forms readily from 5’,5’’’-P1,P4-tetraphosphate in the presence of phosphate-based adenylate traps via a ‘reverse-trap’ mechanism (5’,5’’’-P1,P4-tetraphosphate + diphosphate= 5’,5’’’-P1,P3-triphosphate + phosphate)
deletion of Lig I (PFL13) causes only a slight, 1.6fold increase in background Ap4A but has no effect on the level reached after treatment with MMC, indicating that Lig I cannot be responsible for the MMC-induced increase in Ap4A. Lig III, cf. EC 22.214.171.124, is the most likely, if not sole ligase, contributing to MMC-enhanced Ap4A synthesis. Normal NUDT2 expression does appear to limit the extent of Ap4A accumulation after DNA damage, but suppression of the activity of a hydrolytic activity such as the NUDT2 Ap4A hydrolase does not seem to be reponsible for Ap4A increase after DNA damage
non-cytotoxic doses of certain DNA damaging agents increase diadenosine 5',5'''-P1,P4-tetraphosphate, Ap4A, to concentrations that can inhibit the initiation of DNA replication in a mammalian cell-free system and provide some pointers to the mechanism underlying this increase and its function. Accumulation occurs in vivo of ADP-ribosylated derivatives of Ap4A in response to DNA damage. Lig III is the most likely, if not sole ligase, contributing to MMC-enhanced Ap4A synthesis. Lig III-mediated Ap4A synthesis in response to an increased level of unrepaired strand breaks in APTX-deficient cells
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phosphorylated LysU is able to retain activity (70% activity after 7 days) for significantly longer after storage at 4°C than unphosphorylated LysU (30% activity after 7 days), suggesting that phosphorylated LysU is significantly more stable than nonphosphorylated LysU.