Microcephaly family protein MCPH1 stabilizes RAD51 filaments

Abstract

Microcephalin 1 (MCPH1) was identified from genetic mutations in patients with primary autosomal recessive microcephaly. In response to DNA double-strand breaks (DSBs), MCPH1 forms damage-induced foci and recruits BRCA2-RAD51 complex, a key component of the DSB repair machinery for homologous recombination (HR), to damage sites. Accordingly, the efficiency of HR is significantly attenuated upon depletion of MCPH1. The biochemical characteristics of MCPH1 and its functional interaction with the HR machinery had remained unclear due to lack of highly purified MCPH1 recombinant protein for functional study. Here, we established a mammalian expression system to express and purify MCPH1 protein. We show that MCPH1 is a bona fide DNA-binding protein and provide direct biochemical analysis of this MCPH family protein. Furthermore, we reveal that MCPH1 directly interacts with RAD51 at multiple contact points, providing evidence for how MCPH1 physically engages with the HR machinery. Importantly, we demonstrate that MCPH1 enhances the stability of RAD51 on single-strand DNA, a prerequisite step for RAD51-mediated recombination. Single-molecule tethered particle motion analysis showed a ∼2-fold increase in the lifetime of RAD51-ssDNA filaments in the presence of MCPH1. Thus, our study demonstrates direct crosstalk between microcephaly protein MCPH1 and the recombination component RAD51 for DSB repair.

Figure 1.

Expression, purification, and identification of MCPH1 protein. (A) The expression plasmid of MCPH1. (B) The MCPH1 expression plasmid was transfected and expressed in human Expi293F cells. Whole cell extract was analyzed in a 10% SDS-denaturing polyacrylamide gel and stained with Coomassie Blue (stain) or western blot with anti-MCPH1 antibody (blot). (C) Schematic of our purification protocol. The Expi293F cell extract was fractionated through amylose, Ni⁺-NTA, phenyl HP and heparin columns. The MBP tag was removed using GST-tagged PreScission protease and then filtered through a GST column to remove the protease. (D) Purified MCPH1 protein (1 μg), as analyzed in a 12% SDS-denaturing polyacrylamide gel with Coomassie Blue staining. (E) Results from ESI-MS/MS analysis of purified MCPH1. Identified fragments are shaded in gray.

Figure 2.

MCPH1 possesses DNA-binding activity. (A) Indicated amounts of tag-free MCPH1 were incubated with circular Φx174 ssDNA (6 μM nucleotides). (B) or supercoiled Φx174 dsDNA (6 μM base pairs). The DNA species were then revealed in 1% TBE agarose gels and stained with SYBR gold. In lane 6, the reaction mixture was treated with proteinase K and SDS (P/S) to release the DNA from the nucleoprotein complex. To assess oligo DNA binding, indicated amounts of MCPH1 were incubated with isotope-labeled DNA substrates (750 nM nucleotides or base pairs) as follows: (C) ssDNA (Oligo 3, lanes 1–5) or D-loop (Oligo 2 + 3 + 4, lanes 6–10) and (D) ssDNA (Oligo 5, lanes 1–5), dsDNA (Oligo 5 + 9, lanes 6–10), 3′-overhang DNA (Oligo 5 + 10, lanes 11–15) or Holliday junction (Oligo 5 + 6 + 7 + 8, lanes 16–20). The ³²P-label is denoted by the asterisk. The DNA species were revealed in 6% polyacrylamide gels in TBE buffer, the gels were dried, and then analyzed by phosphorimaging. Percentage bound DNA is shown below the gels. Error bars represent the standard deviation (±SD) calculated from three independent experiments.

Figure 3.

MCPH1 physically interacts with RAD51 and DMC1. In an affinity pulldown assay, purified (His)₆-MBP-MCPH1 was pre-incubated with RAD51 (A), DMC1 (B) or RecA (C). The mixtures were then further incubated with Talon resin to capture protein complex via the (His)₆-tag. The supernatant (S), wash (W), and eluate (E) from the reactions were analyzed by means of 12% SDS-denaturing polyacrylamide gels with Coomassie Blue staining. RAD51, DMC1 and RecA alone were included as controls (panels A, B and C; lanes 1–3). (D) Amylose resin was used to pull-down MBP proteins, with RAD51, DMC1 and RecA as negative controls.

Figure 4.

MCPH1 stabilizes RAD51 and DMC1 nucleoprotein filaments. (A) Schematic of our Benzonase protection assay. (B) The 5′-³²P-labeled 80-mer ssDNA (1.5 μM nucleotides) was pre-incubated with RAD51 and then incubated with tag-free MCPH1 (lanes 5–7). RAD51 (lane 3) or MCPH1 alone (lane 4) were included as negative controls. RAD51 with AMP-PNP was included as a positive control. Then, Benzonase was added to challenge the filaments. The reactions were deproteinized and electrophoresed in 10% TBE polyacrylamide gels. The gels were dried and analyzed by phosphorimaging. The ³²P-label is denoted by the asterisk. (C, D) The assay was also conducted using DMC1 (C) or RecA (D) and analyzed in the same way. DMC1 with calcium (0.5 mM) or RecA with ATPγS were included as respective positive controls. Plots of percentage DNA protected are shown below the gels. Error bars represent the standard deviation (±SD) calculated from three independent experiments.

Figure 5.

MCPH1 prevents both RAD51- and DMC1-ssDNA filament disassembly. (A) Schematic of our tethered particle motion disassembly experiments. The surface-bound DNA with bead was pre-incubated with RAD51-ATP or DMC1-ATP to form nucleoprotein filaments. Free recombinases were washed away and then tag-free MCPH1 was added. Disassembly of recombinase-ssDNA filaments was monitored in real-time by characterizing the change in time-lapsed Brownian motion (BM) of the DNA tethers. (B, C) Representative BM time traces of RAD51–ssDNA filament disassembly in the absence of MCPH1 (B) and in the presence of 25 nM MCPH1 (C). (D) Survival probability histograms of RAD51–ssDNA filament lifetime at different MCPH1 concentrations. AMP-PNP was included to replace ATP for RAD51 as a positive control. Fitted curves are based on the Kaplan-Meier estimator. (E) Bar graph of RAD51–ssDNA filament lifetime obtained from D. (F, G) Representative BM time traces of DMC1-ssDNA filament disassembly in the absence of MCPH1 (F) and in the presence of 25 nM MCPH1 (G). (H) Survival probability histograms of DMC1-ssDNA filament lifetime under different conditions. (I) Bar graph of DMC1-ssDNA filament lifetime compiled from (H). Data include at least 32 DNA tethers from at least three independent experiments. Error bars represent the standard deviation (±SD). Symbol meaning: *P ≤ 0.05; **P ≤ 0.01.