Biology subjects

Hayward, S. B.

Publications and source records attributed to Hayward, S. B..

3 recordsLinked to original sources

bioRxiv2026

Mapping the genetic landscape of the DNA damage response with Cas12a-based combinatorial knockout screens

The DNA damage response (DDR) is a complex network of cellular pathways that ensures the faithful maintenance of our genomes upon a wide array of genomic insults. To elucidate the functional architecture of this network, we conducted unbiased genetic interaction screens using the Cas12a genome editor to disrupt 233 DDR genes frequently mutated in cancer and other genetic diseases, either individually or in pairwise combinations. This approach enabled us to assess the phenotypic effects induced by the disruption of >27,000 DDR gene pair combinations under unperturbed cell growth conditions. From this analysis, we identified over 750 high-confidence positive (buffering) or negative (synthetic lethal/sick) gene-gene interactions, along with multiple connections between previously unlinked DDR pathways and modules, allowing us to define novel aspects of the cellular response to spontaneous, DNA replication-associated DNA damage. Among the identified genetic interactions, we uncovered profound synthetic lethal interactions between genes encoding 1) the translesion polymerase REV1-Pol {zeta} complex and the MCM8-MCM9-HROB DNA helicase complex; 2) Fanconi Anemia (FA) proteins and the mitotic DNA repair factors GEN1, CIP2A, and RHINO; and 3) the DNA translocase SMARCAL1 and components of the FANCM complex, suggesting novel opportunities for targeted therapies in tumors carrying mutations in these genes. Additionally, we identified robust suppressor interactions between the DCLRE1B gene encoding the nuclease APOLLO and the core non-homologous end joining (NHEJ) genes XRCC4, LIG4, and NHEJ1, suggesting that NHEJ impairs the fitness of APOLLO-deficient cells. This work provides a functional map of the DDR network and demonstrates the power of Cas12a-based screens for identifying synthetic lethal and buffering interactions with therapeutic potential.

genetics↗

bioRxiv2025

Mechanistic Insights into Proteomic Mutation-Phenotype Linkages from Tiling Mutagenesis Screens

High-throughput mutagenesis screens are powerful tools for mapping mutations to phenotypes. However, deciphering the molecular mechanisms that link mutations to phenotypic outcomes remains a significant challenge. Here, we present ProTiler-Mut, a versatile computational framework that harnesses tiling mutagenesis screens, which introduce variants across entire protein sequences, to facilitate investigation of mutation-to-phenotype associations at multiple levels, including individual residues, protein substructures, and protein-protein interactions (PPIs). As demonstrated through our analyses of base editing (BE) screens targeting DNA Damage Response (DDR) proteins and T cell regulators, ProTiler-Mut provides novel insights into the mutation-phenotype linkages, including: i) refined classification of mutation that reveals separation-of-function (SOF) category beyond the conventional binary classification of loss-of-function (LOF) and gain-of-function (GOF); ii) definition of phenotype-associated hotspot substructures that enable the inference of the function of unscreened pathogenic mutations; and iii) identification of phenotype-associated PPIs disrupted by functional mutations. Through ProTiler-Mut analyses, we identified a substructure harboring pathogenic GOF mutations that disrupt interactions between the kinases MAPK1 and RSK1, leading to MAPK1 activation and elevated expression of the immune checkpoint receptor PD-1. Furthermore, we demonstrate the applicability of ProTiler-Mut to various mutagenesis screening platforms, highlighting its broad utility and generalizability.

bioinformatics↗

bioRxiv2019

Identification of MCM8IP, an interactor of MCM8-9 and RPA1 that promotes homologous recombination and DNA synthesis in response to DNA damage

Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. HR is carried out by a complex network of DNA repair factors. Here we identify C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a novel DNA repair factor involved in HR. MCM8IP-deficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition. Importantly, we identify a direct interaction with MCM8-9, a putative helicase complex mutated in Primary Ovarian Insufficiency, that is crucial for MCM8IPs ability to promote resistance to DNA damaging agents. In addition to its association with MCM8-9, MCM8IP also binds directly to RPA1. We show that the interactions of MCM8IP with both MCM8-9 and RPA are required to maintain replication fork progression in response to treatment with crosslinking agents. Collectively, our work identifies MCM8IP as a key regulator of DNA damage-associated DNA synthesis during DNA recombination and replication.

genetics↗