Biology subjects

Heyza, J. R.

Publications and source records attributed to Heyza, J. R..

5 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

The PST repeat region of MDC1 is a tunable multivalent chromatin tethering domain

DNA double strand breaks (DSBs) are widely considered the most cytotoxic DNA lesions occurring in cells because they physically disrupt the connectivity of the DNA double helix. Homologous recombination (HR) is a high-fidelity DSB repair pathway that copies the sequence spanning the DNA break from a homologous template, most commonly the sister chromatid. How both DNA ends, and the sister chromatid are held in close proximity during HR is unknown. Here we demonstrate that the PST repeat region of MDC1 is a mutlivalent nucleosome binding domain, sufficient to tether chromatin in multiple contexts. In mitotic cells the affinity of the PST repeats for chromatin is downregulated by phosphorylation to prevent chromosome missegregation, while still contributing to DNA break tethering by the MDC1-TOPBP1-CIP2A complex. In interphase, the PST repeat region is critical for RAD51 focus formation but not the recruitment of 53BP1 to DNA breaks, consistent with a chromatin tethering function. In total, this work demonstrates that the PST repeat region of MDC1 is a multivalent chromatin binding domain with tunable affinity that contributes to DNA break tethering during HR and in mitosis.

cell biology↗

bioRxiv2023

Single-molecule imaging reveals the kinetics of non-homologous end-joining in living cells

Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double-stranded breaks (DSBs) in vertebrates. However, due to challenges in detecting DSBs in living cells, the repair capacity of the NHEJ pathway is unknown. The DNA termini of many DSBs must be processed to allow ligation while minimizing genetic changes that result from break repair. Emerging models propose that DNA termini are first synapsed ~115[A] apart in one of several long-range synaptic complexes before transitioning into a short-range synaptic complex that juxtaposes DNA ends to facilitate ligation. The transition from long-range to short-range synaptic complexes involves both conformational and compositional changes of the NHEJ factors bound to the DNA break. Importantly, it is unclear how NHEJ proceeds in vivo because of the challenges involved in analyzing recruitment of NHEJ factors to DSBs over time in living cells. Here, we develop a new approach to study the temporal and compositional dynamics of NHEJ complexes using live cell single-molecule imaging. Our results provide direct evidence for stepwise maturation of the NHEJ complex, pinpoint key regulatory steps in NHEJ progression, and define the overall repair capacity NHEJ in living cells.

cell biology↗

bioRxiv2023

RHINO restricts MMEJ activity to mitosis

DNA double-strand breaks (DSBs) are toxic lesions that can lead to genome instability if not properly repaired. Breaks incurred in G1 phase of the cell cycle are predominantly fixed by non-homologous end-joining (NHEJ), while homologous recombination (HR) is the primary repair pathway in S and G2. Microhomology-mediated end-joining (MMEJ) is intrinsically error-prone and considered a backup DSB repair pathway that becomes essential when HR and NHEJ are compromised. In this study, we uncover MMEJ as the major DSB repair pathway in M phase. Using CRISPR/Cas9-based synthetic lethal screens, we identify subunits of the 9-1-1 complex (RAD9A-HUS1-RAD1) and its interacting partner, RHINO, as critical MMEJ factors. Mechanistically, we show that the function of 9-1-1 and RHINO in MMEJ is inconsistent with their well-established role in ATR signaling. Instead, RHINO plays an unexpected and essential role in directing mutagenic repair to M phase by directly binding to Polymerase theta (Pol{theta}) and promoting its recruitment to DSBs in mitosis. In addition, we provide evidence that mitotic MMEJ repairs persistent DNA damage that originates in S phase but is not repaired by HR. The latter findings could explain the synthetic lethal relationship between POLQ and BRCA1/2 and the synergistic effect of Pol{theta} and PARP inhibitors. In summary, our study identifies MMEJ as the primary pathway for repairing DSBs during mitosis and highlights an unanticipated role for RHINO in directing mutagenic repair to M phase.

molecular biology↗

bioRxiv2022

Systematic analysis of the molecular and biophysical properties of key DNA damage response factors

Repair of DNA double strand breaks (DSBs) is integral to preserving genomic integrity. Therefore, defining the mechanisms underlying DSB repair will enhance our understanding of how defects in these pathways contribute to human disease and could lead to the discovery of new approaches for therapeutic intervention. Here, we established a panel of HaloTagged DNA damage response factors in U2OS cells which enables concentration-dependent protein labeling. Genomic insertion of the HaloTag at the endogenous loci of the repair factors preserves expression levels and proteins retain proper subcellular localization, foci-forming ability, and functionally support DSB repair. We systematically analyzed total cellular protein abundance, measured recruitment kinetics to DSBs, and defined the diffusion dynamics and chromatin binding by live-cell single-molecule imaging. Our work demonstrates that the Shieldin complex, a critical factor in end joining, does not exist in a preassembled state and Shieldin components are recruited to DSBs with different kinetics. Additionally, live-cell single-molecule imaging revealed the constitutive interaction between MDC1 and chromatin mediated by the PST repeat domain of MDC1. Altogether, our studies demonstrate the utility of single-molecule imaging to provide mechanistic insights into DNA repair, which will serve as a powerful resource for characterizing the biophysical properties of DNA repair factors in living cells.

cell biology↗