Faculty Bibliography

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became a global health crisis after its emergence in 2019. Replication of the virus is initiated by binding of the viral spike (S) protein to human angiotensin-converting enzyme 2 (ACE2) on the target cell surface. Mutations acquired by SARS-CoV-2 S variants likely influence virus-target cell interaction. Here, using single-virus tracking to capture these initial steps, we observe how viruses carrying variant S interact with target cells. Specificity for ACE2 occurs for viruses with the reference sequence or D614G mutation. Analysis of the Alpha, Beta, and Delta SARS-CoV-2 variant S proteins revealed a progressive altered cell interaction with a reduced dependence on ACE2. Notably, the Delta variant S affinity was independent of ACE2. These enhanced interactions may account for the increased transmissibility of variants. Knowledge of how mutations influence cell interaction is essential for vaccine development against emerging variants of SARS-CoV-2.

DNA polymerase epsilon (PolE) in an enzyme essential for DNA replication. Deficiencies and mutations in PolE cause severe developmental abnormalities and cancers. Paradoxically, the catalytic domain of yeast PolE catalytic subunit is dispensable for survival, and its non-catalytic essential function is linked with replicative helicase (CMG) assembly. Less is known about the PolE role in replication initiation in human cells. Here we use an auxin-inducible degron system to study the effect of POLE1 depletion on replication initiation in U2OS cells. POLE1-depleted cells were able to assemble CMG helicase and initiate DNA synthesis that failed shortly after. Expression of POLE1 non-catalytic domain rescued this defect resulting in slow, but continuous DNA synthesis. We propose a model where in human U2OS cells POLE1/POLE2 are dispensable for CMG assembly, but essential during later steps of replication initiation. Our study provides some insights into the role of PolE in replication initiation in human cells.

PARP inhibitors (PARPi) have emerged as promising cancer therapeutics capable of targeting specific DNA repair pathways, but their mechanism of action with respect to PARP1-DNA retention remains unclear. Here, we developed single-molecule assays to directly monitor the retention of PARP1 on DNA lesions in real time. Our study reveals a two-step mechanism by which PARPi modulate the retention of PARP1 on DNA lesions, consisting of a primary step of catalytic inhibition via binding competition with NAD⁺ followed by an allosteric modulation of bound PARPi. While clinically relevant PARPi exhibit distinct allosteric modulation activities that can either increase retention of PARP1 on DNA or induce its release, their retention potencies are predominantly determined by their ability to outcompete NAD⁺ binding. These findings provide a mechanistic basis for improved PARPi selection according to their characteristic activities and enable further development of more potent inhibitors.

BACKGROUND:gene, which encodes the PKP2 protein (plakophilin-2). METHODS:studied at a time of preserved left ventricular ejection fraction and in human induced pluripotent stem cell-derived PKP2-deficient myocytes. RESULTS: CONCLUSIONS:

V(D)J recombination is an essential mechanism of the adaptive immune system, producing a diverse set of antigen receptors in developing lymphocytes via regulated double strand DNA break and subsequent repair. DNA cleavage is initiated by the recombinase complex, consisting of lymphocyte specific proteins RAG1 and RAG2, while the repair phase is completed by classical non-homologous end joining (NHEJ). Many of the individual steps of this process have been well described and new research has increased the scale to understand the mechanisms of initiation and intermediate stages of the pathway. In this review we discuss 1) the regulatory functions of RAGs, 2) recruitment of RAGs to the site of recombination and formation of a paired complex, 3) the transition from a post-cleavage complex containing RAGs and cleaved DNA ends to the NHEJ repair phase, and 4) the potential redundant roles of certain factors in repairing the break. Regulatory (non-core) domains of RAGs are not necessary for catalytic activity, but likely influence recruitment and stabilization through interaction with modified histones and conformational changes. To form long range paired complexes, recent studies have found evidence in support of large scale chromosomal contraction through various factors to utilize diverse gene segments. Following the paired cleavage event, four broken DNA ends must now make a regulated transition to the repair phase, which can be controlled by dynamic conformational changes and post-translational modification of the factors involved. Additionally, we examine the overlapping roles of certain NHEJ factors which allows for prevention of genomic instability due to incomplete repair in the absence of one, but are lethal in combined knockouts. To conclude, we focus on the importance of understanding the detail of these processes in regards to off-target recombination or deficiency-mediated clinical manifestations.

The deubiquitinase USP1 is a critical regulator of genome integrity through the deubiquitylation of Fanconi Anemia proteins and the DNA replication processivity factor, proliferating cell nuclear antigen (PCNA). Uniquely, following UV irradiation, USP1 self-inactivates through autocleavage, which enables its own degradation and in turn, upregulates PCNA monoubiquitylation. However, the functional role for this autocleavage event during physiological conditions remains elusive. Herein, we discover that cells harboring an autocleavage-defective USP1 mutant, while still able to robustly deubiquitylate PCNA, experience more replication fork-stalling and premature fork termination events. Using super-resolution microscopy and live-cell single-molecule tracking, we show that these defects are related to the inability of this USP1 mutant to be properly recycled from sites of active DNA synthesis, resulting in replication-associated lesions. Furthermore, we find that the removal of USP1 molecules from DNA is facilitated by the DNA-dependent metalloprotease Spartan to counteract the cytotoxicity caused by "USP1-trapping". We propose a utility of USP1 inhibitors in cancer therapy based on their ability to induce USP1-trapping lesions and consequent replication stress and genomic instability in cancer cells, similar to how non-covalent DNA-protein crosslinks cause cytotoxicity by imposing steric hindrances upon proteins involved in DNA transactions.

Ryanodine receptor 2 (RyR2) is an ion channel in the heart responsible for releasing into the cytosol most of the Ca²⁺ required for contraction. Proper regulation of RyR2 is critical, as highlighted by the association between channel dysfunction and cardiac arrhythmia. Lower RyR2 expression is also observed in some forms of heart disease; however, there is limited information on the impact of this change on excitation-contraction (e-c) coupling, Ca²⁺-dependent arrhythmias, and cardiac performance. We used a constitutive knock-out of RyR2 in rabbits (RyR2-KO) to assess the extent to which a stable decrease in RyR2 expression modulates Ca²⁺ handling in the heart. We found that homozygous knock-out of RyR2 in rabbits is embryonic lethal. Remarkably, heterozygotes (KO^+/-) show ~50% loss of RyR2 protein without developing an overt phenotype at the intact animal and whole heart levels. Instead, we found that KO^+/- myocytes show (1) remodeling of RyR2 clusters, favoring smaller groups in which channels are more densely arranged; (2) lower Ca²⁺ spark frequency and amplitude; (3) slower rate of Ca²⁺ release and mild but significant desynchronization of the Ca²⁺ transient; and (4) a significant decrease in the basal phosphorylation of S2031, likely due to increased association between RyR2 and PP2A. Our data show that RyR2 deficiency, although remarkable at the molecular and subcellular level, has only a modest impact on global Ca²⁺ release and is fully compensated at the whole-heart level. This highlights the redundancy of RyR2 protein expression and the plasticity of the e-c coupling apparatus.

DNA G-quadruplexes (G4s) are stable, non-canonical DNA secondary structures formed within guanine(G)-rich sequences. While extensively studied in vitro, evidence of the occurrence of G4s in vivo has only recently emerged. The formation of G4 structures may pose an obstacle for diverse DNA transactions including replication, which is linked to mutagenesis and genomic instability. A fundamental question in the field has been whether and how the formation of G4s is coupled to the progression of replication forks. This process has remained undefined largely due to the lack of experimental approaches capable of monitoring the presence of G4s and their association with the replication machinery in cells. Here, we describe a detailed multicolor single-molecule localization microscopy (SMLM) protocol for detecting nanoscale spatial-association of DNA G4s with the cellular replisome complex. This method offers a unique platform for visualizing the mechanisms of G4 formation at the molecular level, as well as addressing key biological questions as to the functional roles of these structures in the maintenance of genome integrity.

Mammalian cells use diverse pathways to prevent deleterious consequences during DNA replication, yet the mechanism by which cells survey individual replisomes to detect spontaneous replication impediments at the basal level, and their accumulation during replication stress, remain undefined. Here, we used single-molecule localization microscopy coupled with high-order-correlation image-mining algorithms to quantify the composition of individual replisomes in single cells during unperturbed replication and under replicative stress. We identified a basal-level activity of ATR that monitors and regulates the amounts of RPA at forks during normal replication. Replication-stress amplifies the basal activity through the increased volume of ATR-RPA interaction and diffusion-driven enrichment of ATR at forks. This localized crowding of ATR enhances its collision probability, stimulating the activation of its replication-stress response. Finally, we provide a computational model describing how the basal activity of ATR is amplified to produce its canonical replication stress response.