Biology subjects

Bielinsky, A. K.

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

2 recordsLinked to original sources

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↗