DNA base excision repair of 5-hydroxymethyl-2'-deoxyuridine stimulates poly(ADP-ribose) synthesis in Chinese hamster cells

Abstract

5-Hydroxymethyl-2'-deoxyuridine (hmdUrd) is incorporated into DNA as a thymidine analog resulting in extensive substitution of thymine residues with 5-hydroxymethyluracil (hmUra) residues. These hmUra residues are then subject to excision by action of hmUra-DNA glycosylase. 3-Aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) synthesis, is toxic to cells that incorporate and repair hmdUrd. To demonstrate that incorporation and repair of hmdUrd stimulates synthesis of poly(ADP-ribose) from intracellular NAD, V79 hamster cells were treated with hmdUrd and intracellular NAD levels were measured. Following hmdUrd treatment, NAD levels fell markedly (80-90%) within 4 h and remained low for at least 10 h, before partially recovering by 24 h. The degree of NAD lowering was dose dependent and paralleled net hmdUrd incorporation. The NAD lowering was largely prevented by concurrent treatment with 4 mM 3AB. No effects on NAD levels were seen following treatment with deoxythymidine or bromodeoxyuridine, which are incorporated into DNA but, in contrast to hmdUrd, are not repaired. When the incorporation of hmdUrd into DNA was blocked with hydroxyurea or aphidicolin, no NAD lowering was seen. HmdUrd also did not produce lowering of NAD concentrations in mutant cell strains deficient in the ability either to incorporate hmdUrd into DNA or to repair hmdUrd from DNA. These results demonstrate that synthesis of poly(ADP-ribose) resulted directly from the incorporation into DNA of the nucleoside hmdUrd and its subsequent repair. These results unequivocally demonstrate that the initiation of normal DNA base excision repair by itself, and not DNA damage per se, is a sufficient stimulus for the induction of poly(ADP-ribose) synthesis.