Poly(ADP-ribose)polymerase 1 stimulates the AP-site cleavage activity of tyrosyl-DNA phosphodiesterase 1

The influence of poly(ADP-ribose)polymerase 1 (PARP1) on the apurinic/apyrimidinic (AP)-site cleavage activity of tyrosyl–DNA phosphodiesterase 1 (TDP1) and interaction of PARP1 and TDP1 were studied. The efficiency of single or clustered AP-site hydrolysis catalysed by TDP1 was estimated. It was shown that the efficiency of AP-site cleavage increases in the presence of an additional AP-site in the opposite DNA strand depending on its position. PARP1 stimulates TDP1; the stimulation effect was abolished in the presence of NAD+. The interaction of these two proteins was characterized quantitatively by measuring the dissociation constant for the TDP1–PARP1 complex using fluorescently-labelled proteins. The distance between the N-termini of the proteins within the complex was estimated using FRET. The data obtained suggest that PARP1 and TDP1 bind in an antiparallel orientation; the N-terminus of the former protein interacts with the C-terminal domain of the latter. The functional significance of PARP1 and TDP1 interaction in the process of DNA repair was demonstrated for the first time. * AP site, : apurinic/apyrimidinic site; BER, : base excision repair; dUMP, : deoxyuridine monophosphate; FAM, : 5′-fluorescein; NHS, : N-hydroxysuccinimide; PAR, : poly(ADP-ribose); PARP1, : poly(ADP-ribose)polymerase 1; ROX, : 5′-rhodamine; TAMRA, : tetramethyl-6-carboxyrhodamine; TDP1, : tyrosyl–DNA phosphodiesterase 1; UDG, : uracil–DNA glycosylase