Abstract
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.
Highlights
Protein–protein interactions play a significant role in DNA repair processes and their regulation, which is very important in the case of the repair of clustered damages, where oxidized bases, apurinic/apyrimidinic (AP) sites and strand breaks are situated within one or two turns of the DNA helix and can be located in both the DNA strands
We have recently found that poly(ADP-ribose)polymerase 1 (PARP1) interacts with AP sites via formation of a Schiff base and possesses weak AP- and 5 -deoxyribose phosphate-lyase activities [4,5]
We have shown that human tyrosyl–DNA phosphodiesterase 1 (TDP1) cleaves AP sites with the formation of the Abbreviations: 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
Summary
Protein–protein interactions play a significant role in DNA repair processes and their regulation, which is very important in the case of the repair of clustered damages (or multiple lesions), where oxidized bases, apurinic/apyrimidinic (AP) sites and strand breaks are situated within one or two turns of the DNA helix and can be located in both the DNA strands. After addition of TDP1 or TDP1 and PARP1 simultaneously to final concentrations of 10 or 500 nM (for radioactive and fluorescent DNA respectively) the reaction mixtures were incubated at 37 ◦C for 30 min.
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