Lim, P. X. | Search bioRxiv

bioRxiv2023

BRCA2 promotes genomic integrity and therapy resistance primarily through its role in homology-directed repair

HighlightsO_LIGap suppression requires BRCA2 C-terminal RAD51 binding in mouse and human cells C_LIO_LIBrca2 heterozygosity in mice results in fork protection and gap suppression defects C_LIO_LIGap suppression mitigates sensitivity to hmdU, but only when HDR is unperturbed C_LIO_LIHDR deficiency is the primary driver of chemotherapeutic sensitivity C_LI eTOC blurbLim et al. report that gap suppression as well as fork protection require BRCA2 stabilization of RAD51 filaments in human and mouse cells but have minimal impact on genome integrity, oncogenesis, and drug resistance. BRCA2 suppression of PRIMPOL-mediated replication gaps confers resistance to the nucleotide hmdU, incorporation of which leads to cytotoxic abasic sites.This effect is diminished when HDR is abrogated. SummaryTumor suppressor BRCA2 functions in homology-directed repair (HDR), protection of stalled replication forks, and suppression of replicative gaps. The relative contributions of these pathways to genome integrity and chemotherapy response are under scrutiny. Here, we report that mouse and human cells require a RAD51 filament stabilization motif in BRCA2 for both fork protection and gap suppression, but not HDR. Loss of fork protection and gap suppression do not compromise genome instability or shorten tumor latency in mice or cause replication stress in human mammary cells. By contrast, HDR deficiency increases spontaneous and replication stress-induced chromosome aberrations and tumor predisposition. Unlike with HDR, fork protection and gap suppression defects are also observed in Brca2 heterozygous mouse cells, likely due to reduced RAD51 stabilization at stalled forks and gaps. Gaps arise from PRIMPOL activity, which is associated with sensitivity to 5-hydroxymethyl-2-deoxyuridine due to the formation of abasic sites by SMUG1 glycosylase and is exacerbated by poly(ADP-ribose) polymerase inhibition. However, HDR deficiency ultimately modulates sensitivity to chemotherapeutics, including PARP inhibitors.

molecular biology↗

bioRxiv2020

Prediction of novel components of bacterial ribosomes by crowd-sourced analysis of RNA sequencing data

Bacterial resistance to antibiotics is a growing problem that is projected to cause more deaths than cancer in 2050. Consequently, novel antibiotics are urgently needed. Since more than half of the available antibiotics target the bacterial ribosomes, proteins that are involved in protein synthesis are thus prime targets for the development of novel antibiotics. However, experimental identification of these potential antibiotic target proteins can be labor-intensive and challenging, as these proteins are likely to be poorly characterized and specific to few bacteria. In order to identify these novel proteins, we established a Large-Scale Transcriptomic Analysis Pipeline in Crowd (LSTrAP-Crowd), where 285 individuals processed 26 terabytes of RNA-sequencing data of the 17 most notorious bacterial pathogens. In total, the crowd processed 26,269 RNA-seq experiments and used the data to construct gene co-expression networks, which were used to identify more than a hundred uncharacterized genes that were transcriptionally associated with protein synthesis. We provide the identity of these genes together with the processed gene expression data. The data can be used to identify other vulnerabilities or bacteria, while our approach demonstrates how the processing of gene expression data can be easily crowdsourced.

bioinformatics↗