Microcephalin/MCPH1 Associates with the Condensin II Complex to Function in Homologous Recombination Repair

Jamie L Wood,Yulong Liang,Kaiyi Li,Junjie Chen

doi:10.1074/jbc.m804080200

Abstract

Microcephalin/MCPH1 is one of the causative genes responsible for the autosomal recessive disorder primary microcephaly. Patients with this disease present with mental retardation and dramatic reduction in head size, and cells derived from these patients contain abnormally condensed chromosomes. MCPH1 contains an N-terminal BRCT and tandem C-terminal BRCT domains. More recently, MCPH1 has been implicated in the cellular response to DNA damage; however, the exact mechanism remains unclear. Here, we report the identification Condensin II as a major MCPH1-interacting protein. MCPH1 and Condensin II interact in vivo, mediated by the CAPG2 subunit of Condensin II binding to a middle domain (residues 376-485) of MCPH1. Interestingly, while Condensin II is not required for the IR-induced G2/M checkpoint, Condensin II-depleted cells have a defect in HR repair, which is also present in MCPH1(-/-)MEFs. Moreover, the Condensin II binding region of MCPH1 is also required for HR function. Collectively, we have identified a novel function of MCPH1 to modulate HR repair through Condensin II, and thereby maintain genome integrity.

Highlights

Three mutations have been described for MCPH1, two premature stop codon mutations (S25X, 427insA) and one missense mutation in the N-terminal BRCT domain (T27R) [3, 8, 9]
Our results revealed several MCPH1-associated proteins; the major associated proteins appeared to be members of the Condensin II complex, which is involved in chromosome condensation [19, 21, 28]
When we per- The N-BRCT, but Not the Condensin II Binding Region, Is formed a data base search, we found that this region contains a Required to Rescue premature chromosome condensation (PCC)—MCPHϪ/Ϫ cells recapitulate the highly conserved stretch of residues but does not have any sim- chromosome condensation phenotype observed in cells ilarity with known protein domains (Fig. 2C), indicating that the interaction between MCPH1 and Condensin II may be con- 5 Y

Summary

EXPERIMENTAL PROCEDURES

Cell Culture, Plasmids, and Antibodies—HeLa and 293T cells were purchased from American Tissue Type Culture (Manassas, VA) and maintained in RPMI 1640 supplemented with 10% bovine serum and 1% penicillin/streptomycin at 37 C in 5% CO2 (v/v). Microcephalin/BRIT1 knock-out mouse embryonic fibroblasts (MEFs) were isolated from E14.5 embryos, and cultured in Dulbecco’s modified Eagle’s medium supplemented with 20% fetal bovine serum and 1% penicillin/streptomycin. SiRNA transfection was carried out using Oligofectamine (Invitrogen) per the manufacturer’s instructions. Full-length MCPH1 or its deletions was cloned into pEF1AHA/FLAG retroviral vector using the gateway system. PEF1AMCPH1 and its deletions were co-transfected with pCL-Amphobac in BOSC23-packaging cell lines to produce virus. Viral particles were collected 48 and 72 h after transfection and subsequently used to infect MEF cell lines. Cells were washed and incubated with primary antibodies diluted in 5% goat serum at 37 °C for 20 min. Cells were washed again 1ϫ in PBS, and secondary antibodies either fluorescein isothiocyanate-conjugated goat anti-mouse IgG or rhodamineconjugated goat anti-rabbit IgG for 20 min at 37 °C. Cells were counterstained with DAPI, washed with PBS, and mounted onto slides with anti-Fade

Images were taken using a Nikon

RESULTS

DISCUSSION