Biology subjects

Stark, J. M.

Publications and source records attributed to Stark, J. M..

9 recordsLinked to original sources

bioRxiv2025

53BP1/RIF1 and DNA-PKcs show distinct genetic interactions with diverse chromosomal break repair outcomes.

DNA double strand breaks (DSBs) are the effective lesion of cancer radiotherapy and induce gene editing. 53BP1 accumulates at DSBs and is implicated in end joining (EJ) repair, but its influence on DSB repair is distinct from canonical non-homologous end joining (C-NHEJ). We sought to define the genetic interplay of 53BP1 with C-NHEJ, focusing on the DNA-PKcs kinase. We examined Cas9 DSBs, which largely generates blunt DSBs, since blunt DSB EJ is dependent on C-NHEJ. Loss of 53BP1 does not affect blunt DSB EJ, but causes a reduction in such repair in DNA-PKcs deficient cells. In contrast, disrupting 53BP1 and DNA-PKcs, both alone and together, has similar effects on the type of deletion mutation (increase in microhomology deletions). We found similar effects on EJ with RIF1 loss, which is a downstream effector of 53BP1. Thus, 53BP1/RIF1 appear to play a backup role for DNA-PKcs during blunt DSB EJ, but function in the same pathway to suppress microhomology deletions. In contrast, 53BP1 and DNA-PKcs function independently to suppress homology-directed repair. Finally, DNA-PKcs kinase inhibition causes marked radiosensitivity, which is not additive with loss of 53BP1 and RIF1. Altogether, 53BP1/RIF1 and DNA-PKcs show distinct genetic interactions with diverse DSB repair outcomes.

molecular biology↗

bioRxiv2024

Distinct functions of PAXX and MRI during chromosomal end joining

A key step of Canonical Nonhomologous End Joining (C-NHEJ) is synapsis of DNA double strand break (DSB) ends for ligation. The DNA-PKcs dimer mediates synapsis in a long-range complex with DSB ends remaining apart, whereas the XLF homodimer can mediate synapsis in both long-range and short-range complexes. Recent structural studies found the PAXX homodimer may also facilitate synapsis in long-range complexes with DNA-PKcs via its interactions with Ku70. Thus, we examined the influence of PAXX in C-NHEJ of chromosomal DSBs, which we compared to another Ku-binding factor, MRI. Using EJ of blunt DSBs with Cas9 reporters as a readout for C-NHEJ, we found that PAXX and/or MRI are dispensable. However, when combined with disruption of DNA-PKcs, particularly with DNA-PKcs kinase inhibition, PAXX becomes important for blunt DSB EJ. In contrast, while DNA-PKcs is also important to suppress short deletion mutations with microhomology, this effect is not magnified with PAXX loss. MRI loss had no effect combined with DNA-PKcs disruption, but becomes important for blunt DSB EJ when combined with disruption of XLF, as is PAXX. Finally, XLF loss causes an increase in larger deletions compared to DNA-PKcs inhibition, which is magnified with combined loss of MRI. Altogether, we suggest that PAXX promotes DSB end synapsis during C-NHEJ in a manner that is partially redundant with DNA-PKcs and XLF, whereas MRI appears to be mainly important in the context of XLF disruption. SIGNIFICANCE STATEMENTCanonical Nonhomologous End Joining (C-NHEJ) is a DNA double strand break repair pathway that can repair diverse types of DNA ends, but is prone to causing genetic mutations. In particular, C-NHEJ can cause insertion or deletion mutations that can cause human disease. Therefore, it is critical to understand the mechanisms of C-NHEJ, including and how various factors within the pathway affect repair outcomes. In this study, we elucidate the role of factors PAXX and MRI during chromosomal EJ and their interplay with the C-NHEJ factors DNA-PKcs and XLF.

genetics↗

bioRxiv2024

Functions of PMS2 and MLH1 important for regulation of divergent repeat-mediated deletions

Repeat-mediated deletions (RMDs) are a type of deletion rearrangement that utilizes two repetitive elements to bridge a DNA double-strand break (DSB) that leads to loss of the intervening sequence and one of the repeats. Sequence divergence between repeats causes RMD suppression and indeed this divergence must be resolved in the RMD products. The mismatch repair factor, MLH1, was shown to be critical for both RMD suppression and a polarity of sequence divergence resolution in RMDs. Here, we sought to study the interrelationship between these two aspects of RMD regulation (i.e., RMD suppression and polar divergence resolution), by examining several mutants of MLH1 and its binding partner PMS2. To begin with, we show that PMS2 is also critical for both RMD suppression and polar resolution of sequence divergence in RMD products. Then, with six mutants of the MLH1-PMS2 heterodimer, we found several different patterns: three mutants showed defects in both functions, one mutant showed loss of RMD suppression but not polar divergence resolution, whereas another mutant showed the opposite, and finally one mutant showed loss of RMD suppression but had a complex effect on polar divergence resolution. These findings indicate that RMD suppression vs. polar resolution of sequence divergence are distinct functions of MLH1-PMS2. HIGHLIGHTSO_LIMLH1-PMS2 suppresses divergent repeat-mediated deletions (RMDs). C_LIO_LIMLH1-PMS2 promotes polar resolution of sequence divergence. C_LIO_LISeveral mutants of MLH1-PMS2 affect both aspects of RMDs. C_LIO_LISome MLH1-PMS2 mutants affect only one aspect of RMDs. C_LIO_LISuppression of RMDs vs. polar resolution of divergence appear distinct. C_LI

molecular biology↗

bioRxiv2023

Arginine shortage induces replication stress and confers genotoxic resistance by inhibiting histone H4 translation and promoting PCNA polyubiquitination

The unique arginine dependencies of cancer cell proliferation and survival creates metabolic vulnerability. Here, we investigate the impact of extracellular arginine availability on DNA replication and genotoxic resistance. Using DNA combing assays, we find that when extracellular arginine is limited, cancer cells are arrested at S-phase and DNA replication forks slow or stall instantly until arginine is re-supplied. The translation of new histone H4 is arginine-dependent and impacts DNA replication and the expression of newly synthesized histone H4 is reduced in the avascular nutrient-poor breast cancer xenograft tumor cores. Furthermore, we demonstrate that increased PCNA occupancy and HLTF-catalyzed PCNA K63-linked polyubiquitination protects arginine-starved cells from hydroxyurea-induced, DNA2-catalyzed nascent strand degradation. Finally, arginine-deprived cancer cells are tolerant to genotoxic insults in a PCNA K63-linked polyubiquitination-dependent manner. Together, these findings reveal that extracellular arginine is the "linchpin" for nutrient-regulated DNA replication. Such information could be leveraged to expand current modalities or design new drug targets against cancer.

cancer biology↗

bioRxiv2021

The DNAPKcs long-range C-NHEJ complex is required for blunt DNA end joining when XLF is weakened

Canonical non-homologous end joining (C-NHEJ) factors can assemble into a long-range (LR) complex with DNA ends relatively far apart that contains DNAPKcs, XLF, XRCC4, LIG4, and the KU heterodimer and a short-range (SR) complex lacking DNAPKcs that has the ends positioned for ligation. Since the SR complex can form de novo, the role of the LR complex (i.e., DNAPKcs) for chromosomal EJ is unclear. We have examined EJ of chromosomal blunt DNA double-strand breaks (DSBs), and found that DNAPKcs is significantly less important than XLF and XRCC4 for such EJ. However, weakening XLF via disrupting interaction interfaces (e.g., disrupting the XLF homodimer interface) causes a marked requirement for DNAPKcs, its kinase activity, and its ABCDE-cluster autophosphorylation sites for blunt DSB EJ. In contrast, other aspects of genome maintenance are sensitive to DNAPKcs kinase inhibition in a manner that is not further enhanced by XLF loss (i.e., suppression of homology-directed repair and structural variants, and IR-resistance). We suggest that DNAPKcs is required to position a weakened XLF in an LR complex that can transition into a functional SR complex for blunt DSB EJ, but also has distinct functions for other aspects of genome maintenance.

molecular biology↗

bioRxiv2021

Intestinal helminth infection transforms the CD4+ T cell composition of the skin

Intestinal helminth parasites can alter immune responses to vaccines, other infections, allergens and autoantigens, indicating effects on host immune responses in distal barrier tissues. We herein show that C57BL/6 mice infected with the strictly intestinal nematode Heligmosomoides polygyrus have impaired capacity to initiate skin immune responses and develop skin-resident memory cells to mycobacterial antigens, both during infection and months after deworming therapy. Surprisingly, and in contrast to a previously noted loss of T cells in peripheral lymph nodes, the skin of worm-infected mice harboured higher numbers of CD4+ T cells compared to skin of uninfected controls. H. polygyrus-specific TH2 cells accumulated during infection and remained after worm expulsion. Accumulation of TH2 cells in the skin was associated with increased expression of the skin-homing chemokine receptors CCR4 and CCR10 on CD4+ T cells in blood and mesenteric lymph nodes draining intestinal tissues, indicating gut-to-skin trafficking of cells. In conclusion, we show that infection by a strictly intestinal helminth has long-term effects on immune cell composition and local immune responses to unrelated antigens in the skin, revealing a novel mechanism for T cell colonization and worm-mediated immunosuppression in this organ.

immunology↗

bioRxiv2021

Laboratory mice with a wild microbiota generate strong allergic immune responses

Allergic disorders are caused by a combination of hereditary and environmental factors. The hygiene hypothesis postulates that early life microbial exposures impede the development of subsequent allergic disease. However, unambiguous evidence that microbes reduce the development of allergic disorders is still lacking. Recently developed wildling mice contain a rich and diverse commensal and encounter a repertoire of microbes typical of the wild, with pathogenic potential. Here, we probed the hygiene hypothesis by comparing the development of allergic inflammation in wildlings to that of genetically identical mice lacking diverse microbial exposure. We find that wildlings develop stronger allergic inflammation in response to house dust mites with allergic T cell responses driven not only by cognate peptide antigens, but also by innate cytokines. In all, the results suggest that high microbial content and diversity potentiates, rather than restricts, allergic immune responses. One sentence summaryStrong allergic inflammation in the face of rich and diverse microbial exposures

immunology↗

bioRxiv2021

Sublingual allergen immunotherapy with recombinant dog allergens prevents airway hyperresponsiveness in a model of asthma marked by vigorous TH2 and TH17 cell responses

Allergy to dogs affects around ten percent of the population in developed countries. Immune therapy of allergic patients with dog allergen extracts has shown limited therapeutic benefit. Herein, we established a mouse model of dog allergy and tested the efficacy of a recombinant protein containing Can f 1, f 2, f 4 and f 6 as a sublingual immune therapy (SLIT). Repeated inhalation of dog extracts induced infiltration of the airways by TH2 cells, eosinophils and goblet cells, reminiscent of the house dust mite (HDM) model of asthma. However, dog allergen extracts also induced robust TH17 cell responses, which was associated with a high neutrophilic infiltration of the airways and promoted airway hyperresponsiveness more potently than HDM allergens. scRNA-Seq analysis of T helper cells responding to dog allergens identified several unique clusters with TH17 cells being hallmarked by the expression of several receptors including IL-17RE. Analysis of T cell receptors also depicted a high frequency of clones that were shared between TH17, TH2 and suppressive Treg cells, indicative of the plasticity of T helper cells in this model. Importantly, prophylactic SLIT reduced airway hyperresponsiveness and type 2-mediated inflammation in this model supporting the use of recombinant allergens in immune therapy.

immunology↗

bioRxiv2020

XLF acts as a flexible connector during non-homologous end joining

Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double strand breaks in vertebrates. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. Here we investigate the role of the intrinsically disordered C-terminal tail of XLF, a critical factor in end synapsis. We demonstrate that the XLF tail along with the Ku binding motif (KBM) at the extreme C-terminus are required for end joining. While the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Single-molecule FRET experiments that observe end synapsis in real-time show that this defect is due to a failure to closely align DNA ends. Our data supports a model in which a single C-terminal tail tethers XLF to Ku while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation.

biochemistry↗