Histone ubiquitination by the DNA damage response factors is required for efficient DNA replication in unperturbed S-phase

The ATM/RNF8/RNF168/53BP1 pathway has been extensively characterized in response to DNA double strand breaks. However, it has remained elusive whether the dramatic phenotypes observed in patients missing key DDR factors (e.g. Ataxia telangiectasia, RIDDLE syndrome) truly reflect defects in DNA double strand breaks signalling and repair, as opposed to other yet-uncharacterized functions of this signalling cascade during unperturbed cell proliferation. To gain insight into alternative roles of the classical DDR factors, we addressed the potential role of RNF168 pathway in a different process of crucial importance for genome integrity, i.e. DNA replication. Combining state-of-the-art approaches to study DNA replication (in collaboration with Massimo Lopes lab) and genome stability, we found that loss of RNF168 leads to reduced replication fork progression and to accumulation of reversed forks (panels A-B), particularly evident at repetitive sequences stalling replication. Slow fork progression depends on MRE11-dependent degradation of reversed forks, implicating RNF168 in reversed fork protection and restart. Consistent with regular nucleosomal organization of reversed forks, the replication function of RNF168 requires H2A ubiquitination (panel C). As this novel function is shared with the key DDR players ATM, RNF8 and 53BP1, we propose that double-stranded ends at reversed forks engage classical DDR factors, suggesting an alternative function of this pathway in preventing genome instability and human disease (Schmid et al, 2018 Mol Cell).

 

Fig 3 Histone ubiquination

 

(A) DNA fiber analysis of control cells (siLUC, 72h) and cells depleted of RNF168, using either an inducible shRNA (+Dox) or an siRNA of a different sequence (siRNF168, 48h and 72h). A labelling scheme and fibers of representative size are shown for each condition in the top left panel. (B) Frequency of reversed replication forks in control (-Dox) and RNF168 depleted (+Dox) U2OS shRNF168 cells as found by transmission electron microscopy. (C) Left: Replication fork progression rate in different U2OS cell lines with Dox-inducible expression of siRNA resistant WT or mutated RNF168, including the R57D mutant, specifically impaired in the ubiquitination of histone H2A (R57D res) and the ubiquitin binding deficient mutant of RNF168 (UBD res). All cell lines were depleted of endogenous RNF168. Right: Representative electron micrograph of a denatured reversed replication fork from U2OS cells depleted of RNF168 using an inducible shRNA (P: Parental duplex, D: Daughter duplexes, R: Regressed arm).