Tyrosyl-DNA phosphodiesterase 1 initiates repair of apurinic/apyrimidinic sites

Abstract

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the hydrolysis of the phosphodiester linkage between the DNA 3′ phosphate and a tyrosine residue as well as a variety of other DNA 3′ damaged termini. Recently we have shown that Tdp1 can liberate the 3′ DNA phosphate termini from apurinic/apyrimidinic (AP) sites. Here, we found that Tdp1 is more active in the cleavage of the AP sites inside bubble-DNA structure in comparison to ssDNA containing AP site. Furthermore, Tdp1 hydrolyzes AP sites opposite to bulky fluorescein adduct faster than AP sites located in dsDNA. Whilst the Tdp1 H493R (SCAN1) and H263A mutants retain the ability to bind an AP site-containing DNA, both mutants do not reveal endonuclease activity, further suggesting the specificity of the AP cleavage activity. We suggest that this Tdp1 activity can contribute to the repair of AP sites particularly in DNA structures containing ssDNA region or AP sites in the context of clustered DNA lesions.

Abacavir, an anti–HIV-1 drug, targets TDP1-deficient adult T cell leukemia

“…To confirm that TDP1 catalytic activity is required for cellular tolerance to ABC, we prepared two mutant TDP1 transgenes [ H263A and H493R ( 13 , 24 )] and a wild-type TDP1 transgene ( 25 ) and ectopically expressed them in MT-2 cells to generate MT-2/ TDP1 H263A , MT-2/ TDP1 H493R , and MT-2/ TDP1 WT cells, respectively (fig. S6, A to C).…”

Poly(ADP-ribose) polymerases covalently modify strand break termini in DNA fragmentsin vitro

“…SVPDE1 (snake venom phosphodiesterase 1) from Crotalus adamanteus was from Worthington (Biochemical Corporation). Human tyrosyl-DNA phosphodiesterase 1 (Tdp1) was purified as described previously (44). The purified human Nudix (nucleoside diphosphate-linked moiety X)-type motif 16 (NUDT16) protein was kindly provided by Dr Ivan Ahel (University of Oxford, U.K.).…”

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

“…Recombinant human TDP1 and PARP1 were expressed in the Escherichia coli system [strain BL21 DE3 (pLys E)] and purified as described previously ([ 7 , 19 ] and [ 20 ] respectively). The recombinant plasmid pET 16B-TDP1 was kindly provided by Dr K.W.…”

“…Recently, we have shown that human tyrosyl–DNA phosphodiesterase 1 (TDP1) cleaves AP sites with the formation of the 3′- and 5′-phosphate termini, suggesting a novel AP-endonuclese independent pathway of AP-site processing [ 6 – 9 ] that can be involved in the repair of AP sites clustered with bulky DNA lesions [ 7 ]. The unique enzymatic activity of TDP1 removes the covalently-linked adducts of DNA topoisomerase I (Top1) from the DNA 3′-end [ 10 – 12 ].…”