Figure 1.
Effects of RNF-113 depletion on C. elegans survival after interstrand DNA crosslinking (ICL) and the intracellular localization of RNF-113.
(A) Comparison of embryonic hatching rates after knocking down RNF-113 expression with and without ICL treatment. rnf-113 RNAi was performed from the P0 young adult stage by feeding wild-type C. elegans worms E. coli cells expressing double-strand RNA for rnf-113. F1 worms at the L4 stage were treated with TMP (25 µg/ml) plus UVA, and eggs were collected over 24 h and their hatching scored 20 h later. This set of experiments in the wild-type background, was also performed in fcd-2(tm298) and rfs-1(ok1372) mutants. The error bars are SEM (standard errors of the mean). (B) Intracellular localization of RNF-113 in the germ cells of the mitotically proliferating region of C. elegans gonads was detected using polyclonal antibodies against RNF-113. Immuno-staining was performed at 3, 9, 16, and 24 h after ICL treatment in the wild type. The scale bar is 10 µm.
Figure 2.
Focus formation by FCD-2 after ICL treatment is not affected by RNF-113 depletion.
The mitotically proliferating regions of wild-type and rnf-113(RNAi) gonads were immuno-stained using FCD-2 polyclonal antibody at 18 h after ICL (TMP/UVA) treatment. The scale bar is 10 µm.
Figure 3.
RNF-113 depletion attenuates RAD-51 focus formation at ICLs in wild-type worms, but not in rfs-1 mutants.
(A) The mitotically proliferating regions of gonads from wild-type, rnf-113(RNAi), rfs-1(ok1372), and rfs-1(ok1372);rnf-113(RNAi) worms are shown after staining for RAD-51 at 16 h following TMP/UVA treatment. (B) The immuno-staining in (A) was repeated at 3 h after IR (ionizing radiation, 75 Gy) treatment instead of ICL treatment. The scale bars are 10 µm. (C) A focal plane having a maximum number of RAD-51 foci was chosen for each nucleus of germ cells (n = 150 for wild-type and rnf-113(RNAi); n = 30 for rfs-1(ok1372) and rfs-1(ok1372);rnf-113(RNAi)) 16 h after ICL treatment as in (A), and the average numbers of RAD-51 foci per nuclear focal plane are plotted. The error bars are SEM.
Figure 4.
RNF-113 depletion does not affect the formation of RPA-1 foci after ICL treatment, but greatly retards the dissipation of RPA-1 foci.
The mitotically proliferating regions of gonads from wild-type, rnf-113(RNAi), rfs-1(ok1372), and rfs-1(ok1372);rnf-113(RNAi) worms are shown after immuno-staining for RPA-1 at 4 h, 12 h, and 24 h following TMP/UVA treatment. The scale bar is 10 µm.
Figure 5.
RNF-113 has E3 ubiquitin ligase activity in vitro.
Recombinant 6×HIS::RNF-113 was purified from E. coli lysates using Ni-NTA agarose and incubated with E1, HA-ubiquitin, and UbcH5c as E2 enzyme. The reaction products were electrophoresed in two separate 6–18% SDS-polyacrylamide gels and probed with (A) HA and (B) HIS antibodies, respectively.
Figure 6.
Proposed model on the roles of RNF-113 and RFS-1 in loading RAD-51 to replication forks stalled at ICLs.
(A) In the wild-type background, a replication fork stalls spontaneously at an ICL and is cleaved. The resulting one-ended DSB is resected to produce ssDNA (single-strand DNA), to which RPA-1, RFS-1 and an unknown protein ‘X’ bind. RNF-113 is proposed to ubiquitinate X, and the ubiquitinated X together with RFS-1 promotes replacement of RPA-1 on ssDNA by RAD-51. (B) In the absence of RNF-113, X is not ubiquitinated so that RPA-1 cannot be displaced from ssDNA. The presence of RPA-1, together with RFS-1 and unmodified X on ssDNA prevents the loading of RAD-51, and the DNA intermediate is at a dead end. (C) In the absence of RFS-1, RAD-51 cannot be recruited to ssRNA, even though RNF-113 ubiquitinates X on ssDNA. Nevertheless, the DNA intermediate with RPA-1 and ubiquitinated X bound is shunted to an error-prone repair pathway involving nucleotide excision repair (NER) and translesion DNA synthesis (TLS). In the absence of both RNF-113 and RFS-1, the DNA intermediate with RPA-1 and unmodified X is also shunted to the error-prone repair pathway.