Top3-Rmi1 dissolve Rad51-mediated D loops by a topoisomerase-based mechanism

Abstract

The displacement loop (D loop) is a DNA strand invasion product formed during homologous recombination. Disruption of nascent D loops prevents recombination, and during synthesis-dependent strand annealing (SDSA), disruption of D loops extended by DNA polymerase ensures a non-crossover outcome. The proteins implicated in D loop disruption are DNA motor proteins/helicases that act by moving DNA junctions. Here we report that D loops can also be disrupted by DNA topoisomerase 3 (Top3), and this disruption depends on Top3's catalytic activity. Yeast Top3 specifically disrupts D loops mediated by yeast Rad51/Rad54; protein-free D loops or D loop mediated by bacterial RecA protein or human RAD51/RAD54 resist dissolution. Also, the human Topoisomerase IIIa-RMI1-RMI2 complex is capable of dissolving D loops. Consistent with genetic data, we suggest that the extreme growth defect and hyper-recombination phenotype of Top3-deficient yeast cells is partially a result of unprocessed D loops.

Figure 1. Sgs1 disrupts protein-free but not Rad51-mediated D-loops

A, Reaction scheme for deproteinized purified D-loops. B, Purified protein-free D-loops (~1 nM) containing a 5’-end labeled 95-mer were incubated with 0.5 nM Sgs1 or Sgs1hd (Sgs1-K706A), or reaction buffer for 10 min and the reaction products resolved on agarose gels. C, Quantitation of D-loops. Shown are means ± standard deviations of three independent experiments. D, Scheme for Rad51/Rad54-mediated D-loop reaction. E, Representative gel of products from reactions containing 20 nM 5’-end labeled 95-mer, 0.67 µM Rad51 (1 Rad51: 3 nts), 100 nM RPA, 112 nM Rad54, 20 nM supercoiled plasmid DNA, and Sgs1 (0, 1, 5, 10, 20, 50, 100 nM). F, Quantitation of D-loops. Shown are means ± standard deviations of three independent experiments.

Figure 2. Topoisomerase activity is necessary and sufficient for dissolution of Rad51–Rad54 reconstituted D-loops by Sgs1-Top3-Rmi1

A, Reaction scheme and proteins. B, D-loop dissolution by Sgs1, Sgs1-Top3-Rmi1 (STR), Sgs1-K706A-Top3-Rmi1 (DTR), Top3-Rmi1 (TR) (0, 2, 5, 10, 20, 50 nM). C, Quantitation of D-loops. Shown are normalized means ± standard deviations of three independent experiments. The absolute values corresponding to maximal D-loop levels are Sgs1: 14%, DTR: 12%, STR: 11%, and TR: 18%. The Sgs1 data were taken from Figure 1. D, D-loop dissolution by Top3 and Top3cd (Top3-Y356F) (0, 5, 10, 20, 50 nM). E, Quantitation of D-loops. Shown are normalized means ± standard deviations of three independent experiments. The absolute values corresponding to maximal D-loop levels are Top3: 22%, Top3cd: 19%.

Figure 3. Top3-mediated D-loop dissolution is highly specific

Top3 does not dissolve protein-free D-loops. A, Reaction scheme for deproteinized D-loops. B, Deproteinized D-loops (~1 nM) were incubated with 0.5 nM Top3 (T) or Top3-Rmi1 (TR) in the presence or absence of 100 nM RPA. C, Quantitation of D-loops. Shown are normalized means ± standard deviations of three independent experiments. The absolute values corresponding to maximal D-loop levels were buffer 11.9%, Top3 12.6%, and Top3-Rmi1 12%. Top3 does not dissolve RecA-mediated D-loops. D, Reaction scheme for RecA-mediated D-loops. E, RecA D-loop reactions were incubated with 0.5 nM Top3 (T) or Top3-Rmi1 (TR) in the presence or absence of 100 nM yeast RPA (ScRPA) or human RPA (HsRPA). F, Quantitation of D-loops. Shown are normalized means ± standard deviations of three independent experiments. The absolute values corresponding to maximal D-loop levels were buffer (ScRPA 5.8%, HsRPA 7.4%), Top3 (ScRPA 5.5%, HSRPA 6%), and Top3-Rmi1 (ScRPA 5.1%, HsRPA 5.7%). Top3 does not dissolve human RAD51-mediated D-loops. G, Reaction scheme for human RAD51- or RAD51/RAD54-mediated D-loops. H, RAD51 D-loop reactions were incubated with 2 nM Top3 (T) or Top3-Rmi1 (TR) in the presence or absence of 100 nM human RPA (HsRPA). I, Quantitation of D-loops. Shown are normalized means ± standard deviations of three independent experiments. The absolute values corresponding to maximal D-loop levels were buffer (RAD51 7.8%, RAD51/RAD54 7.5%), Top3 (RAD51 7.6%, RAD51/RAD54 7.2%, and Top3-Rmi1 (RAD51 7.1%, RAD51/RAD54 7.1%).

Figure 4. Rmi1 stimulates D-loop dissolution by Top3

A, Reaction scheme for Rmi1-stimulated reactions. B, D-loop dissolution by Top3 or Top3-Rmi1 (0, 2, 5, 10, 20 nM). C, Quantitation of D-loops. Shown are normalized means ± standard deviations of three independent experiments. The absolute values corresponding to maximal D-loop levels are Top3: 21.1%, Top3-Rmi1: 18.7%. D, Reaction scheme with tailed 95-mer. E, D-loop dissolution time course by 2 nM Top3-Rmi1. F, Quantitation of D-loops. Shown are means ± standard deviations of three independent experiments.

Figure 5. Human TOPOIIIα-RMI1–RMI2 dissolves D-loops

A, Reaction scheme for human RAD51/RAD54-mediated D-loops. B, Quantitation of D-loops. The absolute values corresponding to maximal D-loop levels were TR 8 % and TRR 8 %. C, Reaction scheme for yeast Rad51/Rad54-mediated D-loops. D, Quantitation of D-loops. The absolute values corresponding to maximal D-loop levels were TR 12 % and TRR14 %. E, Reaction scheme for deproteinized D-loops. Deproteinized D-loops (~1 nM) were incubated with TR and TRR. F, Quantitation of D-loops. The absolute values corresponding to starting D-loop levels were TR 39 % and TRR 37 %. Shown are normalized means ± standard deviations of three independent experiments.

Figure 6

Different D-loops species during HR-mediated DSB and gap repair. D-loops are a collection of different recombination joint molecules with different DNA junction architecture (3’-end, length, gap invasion) and different HR proteins bound to the individual DNA intermediates. D-loops can form during DSB repair (left) or replication fork-associated gap repair (right) and include nascent D-loops (before extension by DNA polymerase: 3’-end not incorporated or ± branch migration), where proteins involved in strand invasion (e.g. Rad51, Rad51 paralogs, Rad54, RPA, Rad52?, others?) are likely still bound to at least parts of the D-loop (top) and extended D-loops (bottom), where instead or in addition to HR proteins replication proteins (PCNA, RFC, DNA polymerase, RPA) will be present in the D-loop.