BLM and RMI1 alleviate RPA inhibition of TopoIIIα decatenase activity

Abstract

RPA is a single-stranded DNA binding protein that physically associates with the BLM complex. RPA stimulates BLM helicase activity as well as the double Holliday junction dissolution activity of the BLM-topoisomerase IIIα complex. We investigated the effect of RPA on the ssDNA decatenase activity of topoisomerase IIIα. We found that RPA and other ssDNA binding proteins inhibit decatenation by topoisomerase IIIα. Complex formation between BLM, TopoIIIα, and RMI1 ablates inhibition of decatenation by ssDNA binding proteins. Together, these data indicate that inhibition by RPA does not involve species-specific interactions between RPA and BLM-TopoIIIα-RMI1, which contrasts with RPA modulation of double Holliday junction dissolution. We propose that topoisomerase IIIα and RPA compete to bind to single-stranded regions of catenanes. Interactions with BLM and RMI1 enhance toposiomerase IIIα activity, promoting decatenation in the presence of RPA.

Figure 1. RPA inhibits the decatenase activity of both TopoIIIα and EcTop1.

(A) Decatenation reactions containing TopoIIIα (30 nM, lanes 2–4) and RPA (100 nM, lane 3; 200 nM, lanes 4 and 5) as indicated were fractionated on a denaturing polyacrylamide gel and autoradiographed. Quantification of the decatenation products is presented in the histogram, normalized to the reactions in lane 2 (TopoIIIα alone). The percent of catenated substrate converted to circular products is indicated. (B) Decatenation reactions containing EcTop1 (6 nM, lanes 2–4) and RPA (100 nM, lane 3; 200 nM, lanes 4 and 5) as indicated were fractionated on a denaturing polyacrylamide gel and autoradiographed. Quantification of the decatenation products is presented in the histogram, normalized to the reactions in lane 2 (EcTop1 alone). The percent of catenated substrate converted to circular products is indicated.

Figure 2. EcSSB inhibits the decatenase activity of both TopoIIIα and EcTop1.

(A) Decatenation reactions containing TopoIIIα (30 nM, lanes 2–4), EcTop1 (6 nM, lanes 5–7) and EcSSB (100 nM, lanes 3 and 6; 200 nM, lanes 4, 7 and 8) as indicated were fractionated on a denaturing polyacrylamide gel and autoradiographed. Quantification of the decatenation products is presented in the histogram, normalized to the reactions in lane 2 (TopoIIIα alone) or lane 5 (EcTop1 alone). The percent of catenated substrate converted to circular products is indicated. (B) Decatenation reactions containing TopoIIIα (30 nM, lanes 2–5) and EcSSB (0.4 mM, lane 3; 0.8 mM, lane 4; 3.2 mM, lane 5) as indicated were fractionated on a denaturing polyacrylamide gel and autoradiographed. Quantification of the decatenation products is presented in the histogram, normalized to the reactions in lane 2 (TopoIIIα alone). The percent of catenated substrate converted to circular products is indicated.

Figure 3. BLM-RMI1 alleviates RPA inhibition of TopoIIIα decatenase activity.

(A) Decatenation reactions containing TopoIIIα (30 nM, lanes 2–7), RPA (100 nM, lanes 3–8), BLM (33 nM, lane 4; 66 nM, lanes 5 and 9) and RMI1 (100 nM, lane 6; 200 nM, lanes 7 and 10) as indicated were fractionated on a denaturing polyacrylamide gel and autoradiographed. Quantification of the decatenation products is presented in the histogram, normalized to the reactions in lane 2 (TopoIIIα alone). The percent of catenated substrate converted to circular products is indicated. (B) Decatenation reactions containing TopoIIIα (15 nM, lanes 2–8), RPA (100 nM, lanes 3–6, 8 and 9), BLM (17 nM, lanes 4–9), wild type RMI1 (75 nM, lane 5; 150 nM, lanes 6, 7 and 9) and RMI1-LLTD mutant (150 nM, lanes 8 and 10) as indicated were fractionated on a denaturing polyacrylamide gel and autoradiographed. Quantification of the decatenation products is presented in the histogram, normalized to the reactions in lane 2 (TopoIIIα alone). The percent of catenated substrate converted to circular products is indicated. (C) Decatenation reactions containing TopoIIIα (7.5 nM, lanes 2–4), BLM (8 nM, lanes 2–4), RMI1 (38 nM, lanes 2–4) and RPA (140 nM, lane 3; 280 nM, lane 4) as indicated were fractionated on a denaturing polyacrylamide gel and autoradiographed. Quantification of the decatenation products is presented in the histogram, normalized to the reactions in lane 2 (TopoIIIα-BLM-RMI1). The percent of catenated substrate converted to circular products is indicated.

Figure 4. BLM alleviates EcSSB inhibition of TopoIIIα, but not EcTop1, decatenase activity.

(A) Decatenation reactions containing TopoIIIα (30 nM, lanes 2–8), EcSSB (3.2 mM, lanes 3–9), BLM (33 nM, lanes 4, 8 and 9; 66 nM, lane 5) and RMI1 (100 nM, lanes 6, 8 and 9; 200 nM, lane 7) as indicated were fractionated on a denaturing polyacrylamide gel and autoradiographed. Quantification of the decatenation products is presented in the histogram, normalized to the reactions in lane 2 (TopoIIIα alone). The percent of catenated substrate converted to circular products is indicated. (B) Decatenation reactions containing TopoIIIα (20 nM, lanes 2–9), RPA (200 nM, lanes 3–5 and 10), EcSSB (3.2 mM, lanes 6–8 and 10) and BLM (33 nM, lanes 4 and 7; 66 nM, lanes 5 and 8–10) as indicated were fractionated on a denaturing polyacrylamide gel and autoradiographed. Quantification of the decatenation products is presented in the histogram, normalized to the reactions in lane 2 (TopoIIIα alone). The percent of catenated substrate converted to circular products is indicated. (C) Decatenation reactions containing EcTop1 (6 nM, lanes 2–7), EcSSB (100 nM, lanes 3–6 and 8), BLM (17 nM, lanes 4–8) and RMI1 (75 nM, lane 5; 150 nM, lanes 6–8) as indicated were fractionated on a denaturing polyacrylamide gel and autoradiographed. Quantification of the decatenation products is presented in the histogram, normalized to the reactions in lane 2 (EcTop1 alone). The percent of catenated substrate converted to circular products is indicated.

Figure 5. TopoIIIα, BLM and RMI1 cooperate to catalyze decatenation on RPA-coated single-stranded catenane.

(A) In a competitive binding model, TopoIIIα alone is unable to access RPA-coated substrate. (B) Complex formation by BLM, TopoIIIα, and RMI1 enables displacement of RPA from the substrate. (C) Alternatively, the DNA binding activity of BLM drives the complex to the substrate even when the substrate is coated with RPA, promoting decatenation.