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. 2012 Aug;94(8):1749-53.
doi: 10.1016/j.biochi.2012.04.004. Epub 2012 Apr 12.

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

Natalia A Lebedeva  1 , Nadejda I Rechkunova, Sherif F El-Khamisy, Olga I Lavrik
Affiliations

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

Natalia A Lebedeva et al. Biochimie. 2012 Aug.

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.

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Figures

Fig. 1
Fig. 1
Schematic view of DNA structures and nucleotide analog used in the experiments.
Fig. 2
Fig. 2
Kinetics of Tdp1 cleavage activity on different AP-DNA substrates. The AP-DNA substrates were treated from 1 to 40 min with 100 nM Tdp1 after incubation DNA with UDG (0.5 U/μl) for 15 min at 37 °C. The percent conversion from the substrate to repair products was calculated. The experiment was performed in triplicate, and the standard deviation for each point is indicated by error bars. Closed circles, AP-DNA/bubble; closed squares, ssAP-DNA; closed triangles, AP-DNA/Flu; closed rhombus, dsAP-DNA.
Fig. 3
Fig. 3
The influence of AP site position in DNA structures on the Tdp1 activity. Graph of Tdp1 activity measured as the percentage of AP-DNA substrates cleaved to product. Defined amounts of Tdp1 (100 nM) were incubated for 30 min with 10 nM ssDNA substrate or dsDNA duplex containing AP site.
Fig. 4
Fig. 4
Repair of AP site initiated by Tdp1 in DNA duplexes containing AP site in cluster with bulky lesion. The 5′-end labeled AP-DNA substrate was subsequently incubated with the UDG (lane 2), Tdp1 (lane 3), PNKP and XRCC1 (lane 4), Pol β (lane 5), DNA ligase III (lane 6) to monitor DNA repair. Lane 1 corresponds to the AP-DNA. The components present in different reaction mixtures are indicated.
Fig. 5
Fig. 5
Binding and AP site cleavage by Tdp1 and its mutants. The A panel shows quantitative analysis of the data from three independent of gel shift experiments. The 5′-end labeled DNA substrates (ssAP-DNA or dsAP-DNA) were incubated with increasing concentrations of Tdp1 or its mutants (from 1 nM up to 10 nM) and subjected to native gel electrophoresis. The gels were dried and analyzed using the PhosphorImager. The B panel shows the hydrolysis of AP sites in single (lane 7–12) and double (lane 1–6) stranded DNAs by Tdp1 and its mutants. The 5′-end labeled AP-DNA was incubated with decreasing concentrations of Tdp1 (lanes 1, 2, 7, 8), SCAN1 (lanes 3, 4, 9, 10), H263A (lanes 5, 6, 11, 12). S denotes the initial substrate, the 48-mer DNA duplex.
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