Biology subjects

Bielinsky, A. K.

Publications and source records attributed to Bielinsky, A. K..

3 recordsLinked to original sources

bioRxiv2024

Cell type specific suppression of hyper-recombination by human RAD18 is linked to PCNA K164 ubiquitination

Homologous recombination (HR) and translesion synthesis (TLS) promote gap-filling DNA synthesis to complete genome replication. One factor involved in both pathways is RAD18, an E3 ubiquitin ligase. Although RAD18s role in promoting TLS through the ubiquitination of PCNA at lysine 164 (K164) is well established, its requirement for HR-based mechanisms is currently less clear. To assess this, we inactivated RAD18 in three human cell lines. Our analyses found that loss of RAD18 in HCT116, but neither hTERT RPE-1 nor DLD1 cell lines, resulted in elevated sister chromatid exchange, gene conversion, and gene targeting, i.e. HCT116 mutants were hyper-recombinogenic (hyper-rec). Loss of RAD18 also impaired TLS activity in HCT116 cells, but unexpectedly, did not reduce clonogenic survival. Interestingly, these phenotypes appear linked to PCNA K164 ubiquitination, as HCT116 PCNAK164R/+ mutants were also hyper-rec and showed reduced TLS activity, consistent with previous studies in rad18-/-or pcnaK164R avian DT40 mutant cells. Importantly, knockdown of UBC9 to prevent PCNA K164 SUMOylation did not affect hyper-recombination, strengthening the link between increased recombination and RAD18-catalyzed PCNA K164 ubiquitination, but not K164 SUMOylation. Taken together, these data suggest that the roles of human RAD18 in directing distinct gap-filling DNA synthesis pathways varies depending on cell type and that these functions are linked to PCNA ubiquitination.

molecular biology↗

bioRxiv2022

Msh2-Msh3 interferes with DNA metabolism in vivo

Mismatch repair (MMR) is a highly conserved DNA repair pathway that safeguards the genome from errors in DNA replication. In Saccharomyces cerevisiae, two MutS homolog (Msh) complexes, Msh2-Msh3 or Msh2-Msh6, initiate MMR. Msh2-Msh3, the focus of this study, recognizes and directs repair of insertion/deletion loops (IDLs) up to ~17 nucleotides. Msh2-Msh3 also recognizes and binds distinct looped and branched DNA structures with varying affinities, thereby contributing to genome stability outside post-replicative MMR through homologous recombination, double-strand break repair (DSBR), and the DNA damage response. Msh2-Msh3 also promotes genome instability through trinucleotide repeat (TNR) expansions. This non-canonical activity is likely an unfortunate consequence of Msh2-Msh3s intrinsic ability to bind a wide range of DNA structures, including those formed with single-stranded (ss) TNR sequences. We previously demonstrated that Msh2-Msh3 binding to 5 ssDNA flap structures interfered with the in vitro binding and cleavage activities of the flap endonuclease Rad27 (Fen1 in mammals), which promotes 5 ssDNA flap processing during Okazaki fragment maturation (OFM) and long-patch base excision repair (LP-BER). Here we demonstrate that elevated Msh2-Msh3 levels interfere with DNA replication and LP-BER in vivo, consistent with the hypothesis that protein abundance and Msh3 ATPase activities are key drivers of Msh2-Msh3-mediated genomic instability.

genetics↗

bioRxiv2020

PCNA-K164 ubiquitination facilitates origin licensing and mitotic DNA synthesis

Ubiquitination of the replication clamp proliferating cell nuclear antigen (PCNA) at the conserved residue lysine 164 (K164) occurs during normal S phase progression and increases after DNA damage induced replication stress. PCNA-K164 ubiquitination is critical for Okazaki fragment maturation and the activation of DNA damage tolerance pathways. Moreover, ubiquitinated PCNA operates in a fork protection pathway parallel to BRCA-RAD51. Whether PCNA ubiquitination regulates other genome maintenance mechanisms is unclear. Utilizing PCNAK164R cells generated by CRISPR-Cas9, we demonstrate that this mutation causes DNA replication defects that impact origin activation. PCNAK164R cells accumulate single-stranded DNA gaps during replication that persist throughout mitosis due to compromised mitotic DNA synthesis (MiDAS). We uncover a novel role for PCNA-K164 ubiquitination in regulating FANCD2 to initiate MiDAS. Persistent gaps hence interfere with MCM2-7 double hexamer loading in the subsequent G1 phase. Our findings demonstrate that the impact of PCNAK164-Ub is not limited to S/G2 phases but extends to mitosis and G1 phase. SUMMARYPCNA-K164 ubiquitination promotes DNA gap filling during S/G2 phases of the cell cycle. This study identifies a novel role for K164 ubiquitination in replication dynamics and mitotic DNA synthesis and thus provides new insight into the players involved in counteracting under-replication.

molecular biology↗