Dss1 regulates interaction of Brh2 with DNA

Abstract

Brh2, the BRCA2 homologue in Ustilago maydis, plays a crucial role in homologous recombination by controlling Rad51. In turn, Brh2 is governed by Dss1, an intrinsically disordered protein that forms a tight complex with the C-terminal region of Brh2. This region of the protein associating with Dss1 is highly conserved in sequence and by comparison with mammalian BRCA2 corresponds to a part of the DNA binding domain with characteristic OB folds. The N-terminal region of Brh2 harbors a less-defined but powerful DNA binding site, the activity of which is revealed upon deletion of the C-terminal region. Full-length Brh2 complexed with Dss1 binds DNA slowly, while the N-terminal fragment binds quickly. The DNA binding activity of full-length Brh2 appears to correlate with dissociation of Dss1. Addition of Dss1 to the heterotypic Brh2-Dss1 complex attenuates DNA binding activity, but not by direct competition for the N-terminal DNA binding site. Conversely, the Brh2-Dss1 complex dissociates more quickly when DNA is present. These findings suggest a model in which binding of Brh2 to DNA is subject to allosteric regulation by Dss1.

FIGURE 1

DNA binding assays. A. Binding reactions containing IRD800 ss60mer and Brh2/Dss1 at the concentrations indicated were incubated for 40 min. Samples were either fixed with glutaraldehyde before electrophoresis or else were run without fixation. Lane a-no protein; b-2 nM; c-4 nM; d-10 nM; e-20 nM; f-100 nM; g-200 nM. B. ³²Pss100mer was incubated with Brh2/Dss1 or Brh2^NT at the concentrations indicated in A and samples fixed with glutaraldehyde. C. Reaction mixes with ³²P-ss60mer incubated with Brh2/Dss1 or Brh2^NT at the concentrations indicated in A were passed onto a manifold through a double layer sandwich of nitrocellulose and DEAE-cellulose membrane. D. Quantification of the results in B and C. E. Binding reactions containing 5′-fluorescein-labeled 49mer and Brh2/Dss1 at the indicated concentrations were incubated for 20 min, then anisotropy was determined. F. Sample mixes containing 100 nM Brh2/Dss1 but no DNA were set up. Glutaraldehyde was then added to samples and incubated for the indicated times before initiating the binding reaction by addition of IRD800 ss60mer. After further incubation for 20 min, samples were analyzed by gel electrophoresis.

FIGURE 2

DNA binding rates. Time courses of DNA binding were determined with single-stranded oligonucleotide and Brh2 or Brh2^NT. Aliquots were removed from binding reactions at times indicated, mixed with glutaraldehyde, and the DNA analyzed for mobility shift after electrophoresis. The fraction of DNA bound was quantified from the relative intensity of the free and shifted oligomer and is shown graphically below. A. Brh2 or Brh2^NT at 20 nM. B. Brh2 or Brh2^NT at 100 nM.

FIGURE 3

Dissociation of Brh2/Dss1 complexes. MBP-tagged Brh2 (represented as N-terminal and C-terminal lobes) complexes with His-tagged Dss1(black ball) is mixed with Ni²⁺-NTA beads. A. Brh2/His-Dss1 complex (550 nM) was incubated at 37° in the presence or absence of DNA (130 nM unlabeled ss60mer). At the indicated times (min) Ni²⁺-NTA beads were added to pull down His-Dss1 either free or complexed with Brh2. Protein composition in the supernatant or bound fractions was determined by SDS gel electrophoresis. Protein was visualized by staining with SimplyBlue Safestain (Invitrogen). The time course of dissociation is shown graphically. B. Reactions mixes contained Brh2/His-Dss1 complex (550 nM) and increasing concentrations of DNA (left to right-20, 40, 130, 330, 660 nM) were incubated for 20 min. The zero point for no DNA added was taken from part A.

FIGURE 4

Dss1 is not associated with Brh2 bound to DNA. A. MBP-Brh2 complexed with ³²P-labeled His-PK-Dss1 was added to reaction mixes with or without IRD 800 ss60mer DNA. After 20 min each mix was transferred to a tube containing Ni²⁺-NTA beads (as packed slurry) and held on ice for 10 min. After brief centrifugation the beads were collected and the supernatant containing the unbound protein fraction was then transferred to a tube containing amylose beads. After 10 min, the beads were collected and the bound fractions from both Ni²⁺-NTA (Ni²⁺) and amylose (amy) plus the final supernatant (sup) were analyzed for protein and DNA composition after SDS gel electrophoresis. Brh2 was visualized by protein staining, Dss1 by phosphorimaging, and DNA by fluorescence detection. B. MBP-Brh2 complexed with ³²P-labeled His-PK-Dss1 was added to reaction mixes with or without 3′-biotin-labeled ss60mer DNA containing IRD700-labeled complementary strand as tracer. After incubation for 40 min mixes were transferred to a tube containing streptavidin coated-magnetic particles. After collecting the beads, protein and DNA in the bound and supernatant fractions were analyzed by SDS-gel electrophoresis.

FIGURE 5

Dss1 attenuates DNA-binding activity of Brh2/Dss1 complex. A. To DNA binding reaction mixtures containing IRD800 labeled ss60mer (3.3 nM) and Brh2/His- Dss1, Brh2, or Brh2^NT (100 nM) was added increasing levels of His-Dss1 or His- Dss1^D37A to the lanes as indicated (0.1, 0.2, 0.4, and 1.0 μM, respectively). After incubation for 20 min, DNA binding was determined. B. Survival of dss1 deletion strain expressing Dss1 or Dss1^D37A was determined after a UV dose of 120 J/m².

FIGURE 6

Brh2 N-terminal domain is required for dissociation of Dss1. A. Brh2^CT/His-Dss1 complex (550 nM) was incubated at 37° in the presence or absence of DNA (130 nM unlabeled ss60mer). At the indicated times (min) Ni²⁺-NTA beads were added to pull down His-Dss1 either free or complexed with Brh2 ^CT. Protein composition in the supernatant or bound fractions was determined by SDS gel electrophoresis. Protein was visualized by staining with SimplyBlue Safestain (Invitrogen). The time course of dissociation is shown graphically. B. Reactions mixes contained Brh2^CT/His-Dss1 complex (550 nM) and increasing concentrations of DNA (left to right-20, 40, 130, 330, 660 nM) were incubated for 20 min. C. Reaction mix contained Brh2^CT/His-Dss1 complex (550 nM) and Brh2^NT(550 nM). In this case Brh2^NT was tagged with MBP and His whereas the MBP-tag was removed from Brh2^CT by cleavage with TEV protease. At the times indicated Ni²⁺-NTA beads were added to pull down His-Dss1 and protein composition in the supernatant and bound fractions was determined.

FIGURE 7

DNA binding rate of Brh2 apoprotein. Time course of DNA binding was determined with IRD800-labeled ss60mer (3.3 nM) and 100 nM Brh2 stripped of Dss1 prepared as described in Material and Methods. Aliquots were removed from binding reactions at times (min) indicated, fixed with glutaraldehyde, and the DNA analyzed for mobility shift after electrophoresis. For the zero time point, DNA was added to a Brh2 reaction mixture already containing glutaraldehyde.

FIGURE 8

Reconstitution of the Brh2/Dss1 complex. Reaction mixtures containing Brh2 apoprotein were mixed with His-Dss1. After 1 hr at 4°, Ni²⁺-NTA beads were added, washed, then eluted with SDS sample buffer (80 μL). Aliquots (10 μL) of the supernatant (first wash) and bound fractions were separated by SDS gel electrophoresis. After staining with SimplyBlue Safestain, bands were quantitated using the Odyssey detection platform. Lane a -- supernatant, no Dss1; lane b -- bound, no Dss1; lane c -- supernatant, plus Dss1; lane d -- bound, plus Dss1; lane e -- 12.5% of total Brh2 apoprotein initially added to the reaction. M--size standards.