Fig. 4: Chimeric Mre11/Rad50 harboring MukB SMC hinge instead of Rad50 zinc hook efficiently functions in repairing DNA damages in vivo.
From: Rad50 zinc hook functions as a constitutive dimerization module interchangeable with SMC hinge
a Left: structural comparison of the P. furiosus Rad50 zinc-hook (upper; PDB 1L8D) and the E. coli MukB hinge (lower; PDB 2WMM). The regions shown in magenta and gray were replaced between the S. pombe Rad50 zinc-hook and the E. coli MukB hinge. The N-terminal and C-terminal coiled-coil regions in the zinc-hook structure are colored in cyan and yellow, respectively. Right: (i) the ring and the head-open structures of S. pombe Mre11/Rad50-MukBhinge (−ATP). (ii) Representative images showing Mre11/Rad50-MukBhinge opening the head. Imaging speed: 500 ms per frame. b Fission yeast rad50-MukBhinge cells do not exhibit cell elongation phenotypes. A rad50+ strain (rad50+:PA, HT1697), a rad50-MukBhinge strain (rad50-MukBhinge:PA, HT1683), and a rad50 deletion strain (∆rad50, HT1250) were grown in YES, followed by observation with DIC microscopy. The scale bar: 5 µm. c Rad52 foci do not increase in rad50-MukBhinge cells. Formation of Rad52 foci was monitored in the following strains carrying the rad52+:YFP allele: rad50+ (rad50+:PA, HT1792), rad50-MukBhinge (rad50-MukBhinge:PA, HT1791), and ∆rad50 (∆rad50, HT1790). Rad52-YFP projection images that are superimposed on bright field microscopy images are presented. The scale bar: 5 µm. d Fission yeast rad50-MukBhinge strains grow as well as wild-type strains in the presence of genotoxins. Sensitivities to CPT and HU were examined in the following strains: an untagged rad50+ wild-type strain (KS1598), two PA-epitope tagged rad50+ strains (HT1633, HT1697), two PA-epitope tagged rad50-MukBhinge strains (HT1655, HT1683), and a ∆rad50 deletion strain (HT1250). e γH2A levels are normal in fission yeast cells carrying the rad50-MukBhinge. γH2A levels were examined by immunoblotting using strains of the following genotypes: rad3+ rad50+ (KS1598), ∆rad3 rad50+ (FY32725), ∆rad3 rad50+:PA (HT1770), ∆rad3 rad50-MukBhinge:PA (HT1768), and ∆rad3 ∆rad50 (HT1782). Source data are provided as a source data file. f rad50-MukBhinge homozygotes sporulate with no visible defect. Mating, meiosis, and sporulation were induced between the following pairs of strains of the opposite mating types: rad50+:PA (HT1693, HT1697), rad50-MukBhinge:PA (HT1680, HT1683), and ∆rad50 (HT1250, HT1257). Sporulation was observed in DIC microscopy. The scale bar: 5 µm.
