Biology subjects

Salguero, I.

Publications and source records attributed to Salguero, I..

4 recordsLinked to original sources

bioRxiv2024

Cytoplasmic localization of ribonucleotide reductase is essential for genome stability

Ribonucleotide reductase (RNR) is a major determinant of dNTP concentrations and its correct regulation is crucial for genome stability. Active RNR is cytoplasmic but the significance of this localization, away from DNA synthesis, is unclear, particularly as RNR is targeted to the nucleus after DNA damage occurs. We subverted the localization of fission yeast RNR by tagging the large (Cdc22R1) and small (Suc22R2) subunits with nuclear localization signal sequences. Directing RNR to the nucleus caused a slow S-phase and increased mutagenesis, in spite of predicted constitutive activation of RNR. Defective function of nuclear RNR is also suggested by the formation of Cdc22R1 supramolecular filaments and defective RNR reoxidation. In contrast, constitutively nuclear Cdc22 or Suc22 has little effect on DNA damage repair and in fact suppresses rad3{Delta}-sensitivity to DNA damaging agents. We suggest that there is an inadequate flux of substrate through nuclear RNR for S phase, but nuclear targeting of RNR may facilitate DNA damage repair under some circumstances.

cell biology↗

bioRxiv2024

A comprehensive genetic catalog of human double-strand break repair

The analysis of DNA sequence outcomes provides molecular insights into double-strand break (DSB) repair mechanisms. By employing parallel in-pool profiling of Cas9-induced indels within a genome-wide knockout library, we present a comprehensive catalog detailing how virtually every human gene influences the DSB repair process. This REPAIRome resource is validated through the identification of novel mechanisms, pathways and factors involved in DSB repair, including unexpected opposing roles for XLF and PAXX in DNA end processing, a molecular explanation for Cas9-induced multi-nucleotide insertions, the identification of HLTF as a DSB-repair factor, the involvement of the SAGA complex in microhomology-mediated end joining, and importantly, an indel mutational signature linked to VHL loss, renal carcinoma and hypoxia. Collectively, these results exemplify the potential of REPAIRome to drive future discoveries in DSB repair, CRISPR-Cas gene editing and the etiology of cancer mutational signatures.

molecular biology↗

bioRxiv2022

MDC1 counteracts restrained replication fork restart and its loss causes chemoresistance in BRCA1/2-deficient mammary tumors

MDC1 is a key protein in DNA damage signaling. When DNA double-strand breaks (DSBs) occur, MDC1 localizes to sites of damage to promote the recruitment of other factors, including the 53BP1-mediated DSB repair pathway. By studying mechanisms of poly(ADP-ribose) polymerase inhibitor (PARPi) resistance in BRCA2;p53-deficient mouse mammary tumors, we identified a thus far unknown role of MDC1 in replication fork biology. MDC1 localizes at active replication forks during normal fork replication and its loss reduces fork speed. We show that MDC1 contributes to the restart of replication forks and thereby promotes sensitivity to PARPi and cisplatin. Loss of MDC1 causes MRE11-mediated resection, resulting in delayed fork restart. This improves DNA damage tolerance and causes chemoresistance in BRCA1/2-deficient cells. Hence, our results show a role for MDC1 in replication fork progression that mediates PARPi- and cisplatin-induced DNA damage, in addition to its role in DSB repair.

molecular biology↗

bioRxiv2020

Mutagenic mechanisms of cancer-associated DNA polymerase ε alleles

A single amino acid residue change in the exonuclease domain of human DNA polymerase {varepsilon}, P286R, is associated with the development of colorectal cancers, and has been shown to impart a mutagenic phenotype. Perhaps unexpectedly, the corresponding Pol {varepsilon} allele in the yeast Saccharomyces cerevisiae (pol2-P301R), was found to drive greater mutagenesis than exonuclease-deficient Pol {varepsilon} (pol2-4), a phenotype sometimes termed ultra-mutagenesis. By studying the impact on mutation frequency, type, replication-strand bias, and sequence context, we show that ultra-mutagenesis is commonly observed in cells carrying a range of cancer-associated Pol {varepsilon} exonuclease domain alleles. Similarities between mutations generated by these alleles and those generated in pol2-4 cells indicate a shared mechanism of mutagenesis that yields a mutation pattern similar to cancer Signature 14. Comparison of POL2 ultra-mutator with pol2-M644G, a mutant in the polymerase domain decreasing Pol {varepsilon} fidelity, revealed unexpected analogies in the sequence context and strand bias of mutations. Analysis of mutational patterns unique to exonuclease domain mutant cells suggests that backtracking of the polymerase, when the mismatched primer end cannot be accommodated in the proofreading domain, results in the observed increase in insertions and T>A mutations in specific sequence contexts.

molecular biology↗