Biology subjects

Vipat, S.

Publications and source records attributed to Vipat, S..

4 recordsLinked to original sources

bioRxiv2025

A new model for coordinating the functions of TIMELESS at the replication fork

TIMELESS is an essential protein that supports a multitude of various cell functions, from replication fork progression through intrinsic barriers in the genome and DNA damage checkpoint to double strand break repair, transcription, and circadian rhythm. How TIMELESS coordinates its various roles at the replication fork and in DNA damage response, and how its canonical position at the leading edge of the replication fork could facilitate its role in DNA damage checkpoint, remain poorly understood. Using an auxin-inducible degron system, we show that TIMELESS-depleted cells exhibited S phase entry defects, and compromised chromatin loading of CLASPIN and TIPIN - the other two components of the Fork Protection Complex (FPC). We further show that FPC chromatin loading was concurrent with the activation of the replicative helicase, but also required proficient DNA synthesis. Proximity labelling experiments suggested the existence of more than one molecule of TIMELESS per replication fork. TIMELESS interaction with the replicative helicase was essential for the speed of replication fork progression, but not for the role of TIMELESS in the activation of the replication checkpoint. Our data propose a new model for coordinating essential functions of TIMELESS in replication fork progression and checkpoint activation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/694947v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@18bb0d7org.highwire.dtl.DTLVardef@71e1aorg.highwire.dtl.DTLVardef@1497a86org.highwire.dtl.DTLVardef@10b0168_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Molecular basis for the host range function of the poxvirus PKR inhibitor E3

The antiviral protein kinase R (PKR) is activated by viral double-stranded RNA and phosphorylates translation initiation factor eIF2, thereby inhibiting translation and virus replication. Most poxviruses contain two PKR inhibitors, called E3 and K3 in vaccinia virus (VACV), which are determinants of viral host range. The prevailing model for E3 function is that it inhibits PKR through the non-specific sequestration of double-stranded (ds) RNA. Our data revealed that Syrian hamster PKR was resistant to E3, which is at odds with the sequestration model. However, Syrian hamster PKR was still sensitive to K3 inhibition. In contrast, Armenian hamster PKR showed opposite sensitivities, being sensitive to E3 and resistant to K3 inhibition. Mutational analyses of hamster PKRs showed that sensitivity to E3 inhibition was largely determined by the region linking the dsRNA-binding domains and the kinase domain of PKR, whereas two amino acid residues in the kinase domain (helix G) determined sensitivity to K3. Expression of PKRs in congenic cells showed that Syrian hamster PKR containing the two Armenian hamster PKR residues in helix-G was resistant to wild type VACV infection, and that cells expressing either hamster PKR recapitulated the phenotypes observed in species-derived cell lines. The observed resistance of Syrian hamster PKR to E3 explains its host range function and challenges the paradigm that dsRNA-binding PKR inhibitors mainly act by the sequestration of dsRNA. SignificanceThe molecular mechanisms that govern the host range of viruses are incompletely understood. A small number of poxvirus genes have been identified that influence the host range of poxviruses. We show that the host range functions of E3 and K3, two host range factors from vaccinia virus, are a result of species-specific interactions with the antiviral protein kinase R (PKR) and that PKR from closely related species displayed dramatic differences in their sensitivities to these viral inhibitors. While there is a substantial body of work demonstrating host-specific interactions with K3, the current model for E3-mediated PKR inhibition is that E3 non-specifically sequesters dsRNA to prevent PKR activation. This model does not predict species-specific sensitivity to E3; therefore, our data suggest that the current model is incomplete, and that dsRNA sequestration is not the primary mechanism for E3 activity.

microbiology↗

bioRxiv2022

The non-catalytic role of DNA polymerase epsilon in replication initiation in human cells

DNA polymerase epsilon in an essential enzyme, responsible for the synthesis of the leading strand during DNA replication. Deficiencies and mutations in DNA polymerase epsilon catalytic subunit (POLE1) cause severe developmental abnormalities and cancers. Paradoxically, the non-catalytic C-terminal domain of yeast polymerase epsilon catalytic subunit (Pol2) is sufficient for cell survival. The non-catalytic essential function of Pol2 in yeast has been associated with its role in the assembly of the replicative helicase CMG. However, the understanding of POLE1 functions in DNA replication initiation in human cells is falling behind. In this study we use an auxin-inducible degron system to study the effect of POLE1 depletion on replication initiation in human cells. Surprisingly, in the absence of POLE1, human cells were able to assemble CMG helicase and initiate DNA synthesis that failed shortly after. Expression of POLE1 C-terminal non-catalytic domain was enough to rescue replication initiation and support slow, but processive DNA synthesis, which was dependent on the POLE1-POLE2 interaction. We propose a model where in human cells POLE1/POLE2 are not essential for CMG assembly, but are required during later steps of replication initiation. Our study provides some insights into the role of DNA polymerase epsilon in replication initiation in human cells.

cell biology↗

bioRxiv2018

Rapid, seamless generation of recombinant poxviruses using host-range and visual selection

Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since this first use as a vaccine, VACV has been developed as a vector for therapeutic vaccines and as an oncolytic virus. These applications take advantage of VACVs easily manipulated genetics and broad host range as an outstanding platform to generate recombinant therapeutics. Several methods have been developed to generate recombinant VACV, including marker selection methods and transient dominant selection. Here, we present a refinement of a host-range selection method coupled with visual identification. Our method takes advantage of selective pressure generated by the host antiviral protein kinase R (PKR) coupled with a fluorescent fusion gene expressing mCherry-tagged E3L, one of two VACV PKR antagonists. This method permits rapid, seamless generation of rVACV in a variety of cell types.

microbiology↗