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.

MCPH1 inhibits Condensin II during interphase by regulating its SMC2-Kleisin interface

“…Previous work has suggested that full-length MCPH1 binds to DNA and chromatin ( Chang et al, 2020 ; Yamashita et al, 2011 ) so we tested whether MBP-MCPH1 1-435 and MBP-MCPH1 196-435 are able to bind to a 50 bp sequence of dsDNA using electrophoretic mobility shift assay (EMSA). Both MCPH1 1-435 -MBP and MCPH1 196-435 -MBP were able to induce a shift, however higher concentrations of MCPH1 196-435 MBP were required for a complete shift in the free DNA band, suggesting MCPH1 1-195 MBP could have a role in DNA binding ( Figure 7B ).…”

“…Binding of MCPH1’s C-terminal BRCT domains to phosphorylated histone H2AX ( Venkatesh and Suresh, 2014 ) is thought to play a part in the DNA damage response, while the premature chromosome condensation caused by mutations within its N-terminal BRCT has been attributed to this domain’s defective association with the SWI/SNF nucleosome remodelling complex and SET1 ( Leung et al, 2011 ). MCPH1 is also thought to bind DNA and has been proposed to function in telomere maintenance, centriole organisation and CHK1 activation ( Alderton et al, 2006 ; Chang et al, 2020 ; Cicconi et al, 2020 ; Gruber et al, 2011 ; Lin and Elledge, 2003 ). In contradiction to the finding that MCPH1 binds directly to condensin II, it is widely believed that the N-terminal ~200 residues of MCPH1 compete with condensin II for chromosomal binding ( Yamashita et al, 2011 ), in other words, MCPH1 has been proposed to occupy loci required for condensin II’s chromosomal activity.…”

ASPM promotes homologous recombination-mediated DNA repair by safeguarding BRCA1 stability

“…Second, MCPH1 interacts with the transcription factor E2F1 and promote its loading on the promoter of BRCA1 to increase the BRCA1 expression ( Yang et al., 2008 ). Last, it has been reported that MCPH1 helps localization of BRCA2 to sites of DSBs and enhances the stability of RAD51-ssDNA filaments, both of which are key players in HR repair ( Wu et al., 2009 ; Chang et al., 2020 ). Thus, ASPM and MCPH1 regulate BRCA1 levels at post-translational stage and transcriptional stage, respectively, in HR repair.…”

MCPH1 inhibits condensin II during interphase by regulating its SMC2-kleisin interface

“…Previous work has suggested that full length MCPH1 binds to DNA and chromatin 33,42 so we tested whether MBP-MCPH11-435 and MBP-MCPH1196-435 are able to bind to a 50 bp sequence of dsDNA using electrophoretic mobility shift assay (EMSA). Both MCPH11-435-MBP and MCPH1196-435-MBP were able to induce a shift, however higher concentrations of MCPH1196-435 MBP were required for a complete shift in the free DNA band, suggesting MCPH11-195 MBP could have a role in DNA binding (Figure 7B).…”

“…Binding of MCPH1's C-terminal BRCT domains to phosphorylated histone H2AX 36 is thought to play a part in the DNA damage response, while the premature chromosome condensation caused by mutations within its N-terminal BRCT has been attributed to this domain's defective association with the SWI/SNF nucleosome remodelling complex and SET1 37 . MCPH1 is also thought to bind DNA and has been proposed to function in telomere maintenance, centriole organisation and CHK1 activation [38][39][40][41][42] . In contradiction to the finding that MCPH1 binds directly to condensin II, it is widely believed that the N-terminal ~200 residues of MCPH1 compete with condensin II for chromosomal binding 33 , in other words, MCPH1 has been proposed to occupy loci required for condensin II's chromosomal activity.…”