Potent inhibition of werner and bloom helicases by DNA minor groove binding drugs

Abstract

Maintenance of genomic integrity is vital to all organisms. A number of human genetic disorders, including Werner Syndrome, Bloom Syndrome and Rothmund-Thomson Syndrome, exhibit genomic instability with some phenotypic characteristics of premature aging and cancer predisposition. Presumably the aberrant cellular and clinical phenotypes in these disorders arise from defects in important DNA metabolic pathways such as replication, recombination or repair. These syndromes are all characterized by defects in a member of the RecQ family of DNA helicases. To obtain a better understanding of how these enzymes function in DNA metabolic pathways that directly influence chromosomal integrity, we have examined the effects of non-covalent DNA modifications on the catalytic activities of purified Werner (WRN) and Bloom (BLM) DNA helicases. A panel of DNA-binding ligands displaying unique properties for interacting with double helical DNA was tested for their effects on the unwinding activity of WRN and BLM helicases on a partial duplex DNA substrate. The levels of inhibition by a number of these compounds were distinct from previously reported values for viral, prokaryotic and eukaryotic helicases. The results demonstrate that BLM and WRN proteins exhibit similar sensitivity profiles to these DNA-binding ligands and are most potently inhibited by the structurally related minor groove binders distamycin A and netropsin (K(i) </=1 microM). The distinct inhibition of WRN and BLM helicases by the minor groove binders suggest that these helicases unwind double-stranded DNA by a related mechanism.

Figure 1

Chemical structures of DNA-binding compounds.

Figure 2

Potent inhibition of WRN and BLM helicase activities on an M13 partial duplex DNA substrate by the minor groove binder distamycin A. WRN protein (96 nM) (A) or BLM protein (19 nM) (B) was incubated with the M13mp18: A-T[5] partial duplex DNA substrate in the presence of the indicated concentrations of distamycin A under the standard helicase reaction conditions as described in Materials and Methods. Incubation was at 24°C for 30 min. Reaction products were analyzed by non-denaturing gel electrophoresis. Representative autoradiographs of WRN and BLM helicase assays are shown. Closed triangle, heat-denatured control; S, no enzyme control. (C) Quantitation of helicase activity (% control activity) as a function of distamycin A concentration. Closed circles, WRN; open circles, BLM. In control reactions, WRN or BLM helicase catalyzed unwinding of ∼50% of the partial duplex substrate. All data points are the average of at least three independent determinations.

Figure 2

Potent inhibition of WRN and BLM helicase activities on an M13 partial duplex DNA substrate by the minor groove binder distamycin A. WRN protein (96 nM) (A) or BLM protein (19 nM) (B) was incubated with the M13mp18: A-T[5] partial duplex DNA substrate in the presence of the indicated concentrations of distamycin A under the standard helicase reaction conditions as described in Materials and Methods. Incubation was at 24°C for 30 min. Reaction products were analyzed by non-denaturing gel electrophoresis. Representative autoradiographs of WRN and BLM helicase assays are shown. Closed triangle, heat-denatured control; S, no enzyme control. (C) Quantitation of helicase activity (% control activity) as a function of distamycin A concentration. Closed circles, WRN; open circles, BLM. In control reactions, WRN or BLM helicase catalyzed unwinding of ∼50% of the partial duplex substrate. All data points are the average of at least three independent determinations.

Figure 2

Potent inhibition of WRN and BLM helicase activities on an M13 partial duplex DNA substrate by the minor groove binder distamycin A. WRN protein (96 nM) (A) or BLM protein (19 nM) (B) was incubated with the M13mp18: A-T[5] partial duplex DNA substrate in the presence of the indicated concentrations of distamycin A under the standard helicase reaction conditions as described in Materials and Methods. Incubation was at 24°C for 30 min. Reaction products were analyzed by non-denaturing gel electrophoresis. Representative autoradiographs of WRN and BLM helicase assays are shown. Closed triangle, heat-denatured control; S, no enzyme control. (C) Quantitation of helicase activity (% control activity) as a function of distamycin A concentration. Closed circles, WRN; open circles, BLM. In control reactions, WRN or BLM helicase catalyzed unwinding of ∼50% of the partial duplex substrate. All data points are the average of at least three independent determinations.

Figure 3

Effect of distamycin A on the ATPase activities of WRN and BLM helicases in the presence of the M13mp18 ssDNA effector. ATPase reactions containing 0.8 mM [³H]ATP, M13mp18 ssDNA (2 µM nucleotide phosphate) and the indicated concentration of distamycin A were initiated with WRN protein (70 nM) (closed circles) or BLM protein (11 nM) (open circles), as described in Materials and Methods. Reactions were incubated at 24°C for 20 min and analyzed for the production of [³H]ADP. In control reactions, WRN or BLM proteins hydrolyzed <20% of the total ATP in the reaction mixture. The total amount of ATP hydrolyzed by WRN and BLM proteins in the absence of distamycin were 1040 and 1140 pmol, respectively. All data points are the average of at least three independent determinations.

Figure 4

Potent inhibition of WRN and BLM helicase activities on an M13 partial duplex DNA substrate with a 4 bp A-T tract by the minor groove binder netropsin. WRN protein (96 nM) or BLM protein (19 nM) was incubated with the M13mp18: A-T[4] partial duplex DNA substrate in the presence of the indicated concentrations of netropsin under the standard helicase reaction conditions described in Materials and Methods. Helicase activity (% control activity) is expressed as a function of netropsin concentration. Closed circles, WRN; open circles, BLM. In control reactions, WRN or BLM helicase catalyzed unwinding of ∼50% of the partial duplex substrate. All data points are the average of at least three independent determinations.

Figure 5

Potent inhibition of WRN and BLM helicase activities on a M13 partial duplex DNA substrate with a 4 bp A-T tract by the minor groove binder distamycin A. WRN protein (96 nM) or BLM protein (19 nM) was incubated with the M13mp18: A-T[4] partial duplex DNA substrate in the presence of the indicated concentrations of netropsin under the standard helicase reaction conditions as described in Materials and Methods. Helicase activity (% control activity) is expressed as a function of distamycin A concentration. Closed circles, WRN; open circles, BLM. In control reactions, WRN or BLM helicase catalyzed unwinding of ∼50% of the partial duplex substrate. All data points are the average of at least three independent determinations.

Figure 6

The presence of hRPA in the WRN or BLM helicase reactions does not alleviate the potent inhibition of unwinding activity by the minor groove binder distamycin A. WRN protein (96 nM) or BLM protein (19 nM) was incubated with the M13mp18: A-T[5] partial duplex DNA substrate in the presence of hRPA (96 nM, heterotrimer) and the indicated concentration of distamycin A. Helicase reactions were conducted as described in Materials and Methods. Helicase activity (% control activity) is expressed as a function of distamycin A concentration. Closed circles, WRN; open circles, BLM. In control reactions, WRN or BLM helicase catalyzed unwinding of ∼50% of the partial duplex substrate. All data points are the average of at least three independent determinations.