Inhibition of BACH1 (FANCJ) helicase by backbone discontinuity is overcome by increased motor ATPase or length of loading strand

Abstract

The BRCA1 associated C-terminal helicase (BACH1) associated with breast cancer has been implicated in double strand break (DSB) repair. More recently, BACH1 (FANCJ) has been genetically linked to the chromosomal instability disorder Fanconi Anemia (FA). Understanding the roles of BACH1 in cellular DNA metabolism and how BACH1 dysfunction leads to tumorigenesis requires a comprehensive investigation of its catalytic mechanism and molecular functions in DNA repair. In this study, we have determined that BACH1 helicase contacts with both the translocating and the non-translocating strands of the duplex are critical for its ability to track along the sugar phosphate backbone and unwind dsDNA. An increased motor ATPase of a BACH1 helicase domain variant (M299I) enabled the helicase to unwind the backbone-modified DNA substrate in a more proficient manner. Alternatively, increasing the length of the 5' tail of the DNA substrate allowed BACH1 to overcome the backbone discontinuity, suggesting that BACH1 loading mechanism is critical for its ability to unwind damaged DNA molecules.

Figure 1

Differential effects of BACH1 polymorphisms on helicase function. Helicase reactions (20 μl) were performed by incubating 4.8 nM BACH1-WT or BACH1 variant (BACH1-M299I, BACH1-P47A) as indicated with 0.5 nM forked duplex DNA substrate (Substrate 1) at 30°C for 15 min in the presence or absence of ATP (2 mM) under standard helicase assay conditions as described under ‘Materials and Methods.’ (A) Lane 1, no enzyme control; Lanes 2–7, indicated BACH1 protein in the presence or absence of ATP; Lane 8, heat-denatured DNA substrate control. A phosphorimage of a typical gel is shown. (B) Quantitative helicase data represent the mean of at least three independent experiments with standard deviation (SD) indicated by error bars, as is the case for the remaining figures. Filled circles, BACH1-WT; open circles, BACH1-M299I; filled squares, BACH1-P47A; open squares, BACH1-K52R.

Figure 2

Effect of polyglycol backbone modifications on BACH1 helicase activity. The indicated concentrations of BACH1-WT or BACH1-M299I were incubated with 0.5 nM DNA substrate containing the polyglycol modification (Substrates 2–4) at 30°C for 15 min under standard helicase assay conditions as described under ‘Materials and Methods.’ Quantitative analyses of BACH1 helicase data are shown. Open triangle, BACH1-WT, Substrate 2; filled circle, BACH1-WT, Substrate 3; cross, BACH1-WT, Substrate 4; filled diamond, BACH1-M299I, Substrate 2; filled square, BACH1-M299I, Substrate 3; open circle, BACH1-M299I, Substrate 4.

Figure 3

Effect of abasic sites on BACH1 helicase activity. 4.8 nM BACH1-WT or 2.4 nM BACH1-M299I was incubated with 0.5 nM DNA substrate containing the three adjacent abasic sites (Panel B, Substrates 2,6,7,8) at >30°C for 15 min under standard helicase assay conditions as described under ‘Materials and Methods.’ Quantitative analyses of BACH1 helicase data are shown.

Figure 4

BACH1-WT is preferentially sequestered by DNA molecules with backbone polyglycol modifications in the duplex region. Sequestration assays with 4.8 nM BACH1-WT and increasing concentrations (0–25 nM) of the indicated forked duplex competitor DNA molecules were performed as described under ‘Materials and Methods.’ Quantitative analyses of the helicase data are shown. Filled diamonds, unmodified forked duplex; filled squares, forked duplex with backbone modification in top (translocating) strand; open circles, forked duplex with backbone modification in bottom (non-translocating) strand; cross, duplex with modification in both strands.

Figure 5

Partial unwinding of backbone modified DNA substrates by BACH1-WT helicase. (A) Schematic of the dye displacement assay to measure helicase activity. The dye molecules are pre-bound to the duplex DNA substrate (dark ovals), causing them to fluoresce. As the dsDNA is unwound, the dye molecules are displaced (open ovals) which is measured by a decrease in the amount of fluorescence. In Scheme 1, the unmodified DNA substrate is fully unwound by the helicase, resulting in a maximal change in fluorescence. In Scheme 2, the backbone modified DNA substrate is partially unwound by the helicase, resulting in a smaller change in fluorescence since a population of dye molecules remain intercalated within the residual duplex. (B) Kinetics of BACH1-WT helicase activity on the unmodified or specified polyglycol modified DNA substrate as measured by the dye-displacement assay. 0.8 nM DNA substrate pre-incubated with 100 nM Hoechst dye was rapidly mixed with 4.6 nM BACH1-WT and 2 mM ATP. Unwinding of the DNA substrate, as shown by percent fluorescence decrease, was measured as a function of time. Red, unmodified Substrate 2; green, Substrate 3 with bottom strand backbone modification; blue, Substrate 4 with top strand backbone modification.

Figure 6

Inhibition of BACH1 helicase activity by the polyglycol backbone modification is overcome by an increased length in the 5 ssDNA tail. 4.8 nM BACH1-WT was incubated with the indicated polyglycol modified forked (35 nt 5 tail, 19 nt 3 tail) duplex [(A) Substrates 9–12] or simple overhang (35 nt 5 tail) duplex [(B) Substrates 13–16] at 30°C for 15 min under standard helicase assay conditions as described under ‘Materials and Methods’. Quantitative analyses of BACH1 helicase data are shown from at least three independent experiments with SD indicated by error bars.

Figure 7

BACH1 protein domains and missense mutations. (A) Cartoon depicting the BACH1 protein with the conserved helicase motifs indicated by red boxes and the positions of the metal binding domain (yellow), nuclear localization sequence (NLS), and BRCA1 interaction domain. BACH1 polymorphic variants (P47A, M299I) are indicated above the protein schematic in blue. Missense mutations (Q255H, A349P, W647C, R707C) genetically linked to Fanconi Anemia (Complementation Group J) are indicated below the protein schematic in pink. (B) Sequence alignment of the region between the Walker A (motif I) and Walker B (motif II) boxes that includes the Fe-S domain for Sulfolobus acidocaldarius XPD (Sac) and a number of human DNA helicases (BACH1 (FANCJ), XPD, RTel1 and Chl1) as adapted from Ref. (39). Conserved cysteine residues of the metal binding domain are highlighted in yellow. The position of a BACH1 amino acid substitution (M299I) adjacent to a conserved cysteine residue and representing a BACH1 polymorphic variant is highlighted in blue. An amino acid substitution (A349P) arising from a mutation genetically linked to Fanconi Anemia (Complementation Group J) and adjacent to a conserved cysteine residue is highlighted in pink.