The role of human Shu complex in ATP-dependent regulation of RAD51 filaments during homologous recombination-associated DNA damage response

Abstract

Error-free DNA lesion bypass is an important pathway in DNA damage tolerance. The Shu complex facilitates this process by promoting homologous recombination (HR) to bypass DNA damage. Biochemical analysis of the human Shu complex homolog, hSWS1-SWSAP1, offers valuable insights into the HR-associated DNA damage response. Here, we biochemically characterized the human Shu complex and examined its interactions with RAD51 filaments, which are essential in HR. Using fluorescence polarization assays, we first revealed that hSWS1-SWSAP1 preferentially binds DNA with an exposed 5' end in the presence of adenine nucleotides. We then investigated and validated the DNA-stimulated ATPase activity of hSWS1-SWSAP1 through site-specific mutagenesis, revealing that DNA with an exposed 5' end is the most efficient in enhancing this activity. Furthermore, we showed that hSWS1-SWSAP1 initially interacts with RAD51 filaments at the 5' end and modulates the properties of the nucleoprotein filaments using fluorescence-based assays. Our findings revealed that hSWS1-SWSAP1 induces conformational changes in RAD51 filaments in an ATP hydrolysis-dependent manner, while its stabilization of the filaments depends on ATP binding. This work provides mechanistic insights into the regulation of RAD51 filaments in HR-associated DNA damage tolerance.

Keywords: ATPase; DNA damage tolerance response; RAD51 filament; Shu complex; homologous recombination.

Figure 1

Purified human Shu complex and its DNA binding.A and B, gel filtration (A) and SDS-PAGE (B) analysis of the purified hSWS1-SWSAP1 (wildtype, WT) and mutants (K18A and D96A in SWSAP1). C, electrophoretic mobility shift assay for WT hSWS1-SWSAP1–DNA interactions. Increasing concentrations of hSWS1-SWSAP1 ([S]) were mixed with 0.05 μM DNA substrates (ss39, ds39, Fork). Protein–DNA mixtures were resolved by 2-layer PAGE gels: 5% native polyacrylamide at the top and 15% at the bottom (dark layer). Assays were performed in triplicate with comparable results (Fig. S3, A–C). LMW, low molecular weight protein ladder.

Figure 2

ATP binding and ATPase activity of hSWS1-SWSAP1.A, fluorescence spectra of TNP-ATP (5 μM) with WT hSWS1-SWSAP1 and Walker motif mutants (K18A, D96A) (4 μM). B, TNP-ATP saturation assay. Increasing concentrations of TNP-ATP were added to hSWS1-SWSAP1 (2 μM). Dissociation constants (K_d^TNP-ATP) were determined by nonlinear curve fitting to a one-site binding model. C, competitions of TNP-ATP (5 μM)/hSWS1-SWSAP1 (4 μM) complexes by ATP or ADP (2.5 mM each). Fluorescence units were normalized to the initial readings without excess ATP and ADP. D, titration of TNP-ATP (2.5 μM) fluorescence with ATP in the presence of hSWS1-SWSAP1 (2 μM). Data shown represent fluorescence units after subtraction of TNP-ATP background fluorescence, followed by normalization to the reading without ATP (relative units). Data were fitted using nonlinear regression to a one-site competitive binding model. Dissociation constant (K_d^ATP) was derived from competitive binding data (see Experimental procedures). E, ATPase activity of WT hSWS1-SWSAP1 and Walker motif mutants (1 μM each) with DNA substrate containing a 5′-overhang (10 μM), over 120-min time course. F, ATPase activity (60-min reactions) of hSWS1-SWSAP1 (1 μM) in the presence of ssDNA and dsDNA substrates with different end types (1 μM each), with increasing ATP concentrations. Data were fitted using the Michaelis–Menten model to determine kinetic parameters (details found in Table 3). Data in A–F represent the mean of three independent replicates, with error bars in B–F indicating the standard deviation from the triplicate experiments. dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; TNP-ATP, 2′(3′)-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate.

Figure 3

hSWS1–SWSAP1 complex enhances DNase I digestion on RAD51 filaments. A–D, 2-hour DNase I digestion of RAD51-ssDNA filaments labeled on (A and C) 5′- or (B and D) 3′-end with 6-FAM and incubated with/without WT hSWS1-SWSAP1 or Walker motif mutants (K18A, D96A). Filaments (2 μM RAD51 on 0.25 μM poly-dT30 ssDNA with 2 mM ATP) were incubated for 10 min and then mixed with increasing concentrations of WT hSWS1-SWSAP1 or mutants ([S]). Digestions were carried out using 2 Unit DNase I at 37 °C. Reaction mixtures were resolved by 22.5% denaturing PAGE. DNA ladder with nucleotide sizes (in nt) is shown to the left of each gel and reused across all panels to indicate the sizes of the substrate and its degradation products. “∗5′-(T)₃₀” and “∗3′-(T)₃₀” stand for 5′- and 3′-labeled poly-dT30 ssDNA, respectively. 6-FAM, 6-carboxyfluorescein; PAGE, polyacrylamide gel electrophoresis; ssDNA, single-stranded DNA.

Figure 4

Conformational and stability changes of RAD51 filaments induced by hSWS1-SWSAP1.A, fluorescence intensity profiles of RAD51-ssDNA filaments with/without hSWS1-SWSAP1 proteins over 60-s time frame. Filaments (1 μM RAD51 on 15 nM 5′- or 3′-labeled poly dT39 ssDNA) were incubated for 10 min and then mixed with WT hSWS1-SWSAP1 or Walker motif mutants (50 nM each), all in the presence of ATP (2 mM). B, fluorescence intensity profiles of RAD51-ssDNA filaments (preincubated with 2 mM ATP) mixed with hSWS1-SWSAP1 (preincubated with 2 mM of the indicated nucleotide), over 60 s. C and D, fluorescence intensity profiles of RAD51-ssDNA filaments with/without WT hSWS1-SWSAP1 and Walker motif mutants, mixed with 100-fold excess of unlabeled ssDNA, over 60 s. Samples were excited at 490 nm, and emission readings were measured at 522 nm at equilibrium (37 °C). Filament-alone data were normalized as the baseline in arbitrary units corresponding to fluorescence intensities (dashed black lines). “∗5′-(T)₃₉” and “∗3′-(T)₃₉” stand for 5′- and 3′-labeled poly-dT39 ssDNA, respectively. 6-FAM, 6-carboxyfluorescein; ssDNA, single-stranded DNA.

Figure 5

Molecular model for the action of hSWS1-SWSAP1 on RAD51-ssDNA filaments. The role of hSWS1-SWSAP1 (hShu) in homologous recombination as a DNA-stimulated ATPase. The hShu complex binds to the 5′ end of a RAD51 filament containing DNA lesions, such as abasic lesions, stabilizing the filament in an ATP-dependent manner. The alteration of the RAD51 filament conformation is dependent on the ATPase activity of hSWS1-SWSAP1, rendering the filament flexible and “open” for HR-associated processes. HR, homologous recombination.