Ectopic RNF168 expression promotes break-induced replication-like DNA synthesis at stalled replication forks

Area of Science:

• DNA damage response
• Ubiquitin ligase function
• Cancer genomics

Background:

• RNF168 is an E3 ubiquitin ligase activated by DNA breaks, ubiquitinating γH2AX.
• RNF168 levels are tightly regulated to control DNA repair and chromatin ubiquitination.
• High RNF168 levels can impede DNA repair and increase sensitivity to PARP inhibitors.

Purpose of the Study:

• To investigate the effect of RNF168 overexpression on hydroxyurea (HU)-induced stalled replication forks in BRCA1-deficient cells.
• To elucidate the molecular mechanisms underlying RNF168-mediated DNA synthesis.
• To determine the role of BRCA1 in regulating RNF168-induced DNA synthesis.

Main Methods:

• Ectopic RNF168 overexpression in BRCA1-deficient cells.
• Hydroxyurea treatment to induce stalled replication forks.
• Analysis of DNA fiber extension and protein recruitment (RAD18, RAD52, POLD3).

Main Results:

• RNF168 overexpression unexpectedly extended DNA fibers in HU-treated BRCA1-deficient cells.
• RNF168 recruited RAD18 to ubiquitinated H2AX at HU-induced breaks.
• A RAD18-SLF1-RAD52-POLD3 axis initiated DNA synthesis, a process blocked by wild-type BRCA1.

Conclusions:

• In BRCA1-deficient cells, supra-physiological RNF168 promotes break-induced replication (BIR)-like DNA synthesis.
• This RNF168-driven BIR may contribute to tandem duplications and mutational signatures in BRCA1-mutated cancers.
• BRCA1 acts as a barrier to RNF168-induced DNA synthesis, highlighting its tumor-suppressive role.