Biology subjects

Tahirov, T. H.

Publications and source records attributed to Tahirov, T. H..

9 recordsLinked to original sources

bioRxiv2026

SOX2 can associate with chromatin directly by binding to DNA or indirectly via association with other chromatin-bound proteins

It is widely assumed that SOX2 regulates gene expression and facilitates the opening of chromatin by binding directly at SOX motifs. To test this assumption, we created a SOX2 DNA binding mutant to determine whether other regions of SOX2 contribute to gene target specificity. When exogenously expressed in cells, this SOX2 mutant [SOX2(G76P)], like elevated unmodified SOX2, dramatically alters the transcriptome, but it does so by regulating vastly different gene sets and gene networks than SOX2. Consistent with their differential effects on the transcriptome, ChIP-seq analysis demonstrates that SOX2 and SOX2(G76P) associate primarily with different genomic loci, and motif analysis indicates that SOX2 binds primarily at SOX motifs, whereas SOX2(G76P) associates with chromatin at non-SOX motifs, including AP-1 motifs. Additionally, ATAC-seq analysis indicates that SOX2 substantially increases chromatin accessibility, but SOX2(G76P) does not. The findings presented lead to the conclusion that SOX2(G76P) associates with chromatin indirectly by a "piggyback" mechanism through its association with other chromatin-associated proteins, including AP-1 complexes. Remarkably, we also show that SOX2 and SOX2(G76P) each associate with a subset of the same gene loci that contain several different DNA motifs, including AP-1 motifs, but no high confidence SOX motifs. Overall, our findings provide new perspectives on SOX2 and lead to two important conclusions: 1) selection of gene targets by SOX2 is not solely determined by its DNA binding domain, and 2) SOX2 not only associates with chromatin directly by binding to SOX motifs but can also associate with a subset of gene loci indirectly through its association with other chromatin-associated proteins.

molecular biology↗

bioRxiv2025

Structural Basis of Herpesvirus Helicase-Primase Inhibition by Pritelivir and Amenamevir

Widespread herpesvirus infections are associated with various diseases. DNA replication of human herpes simplex virus type 1 (HSV-1) requires a helicase-primase (HP) complex of three core proteins: UL5, UL52, and UL8. This complex unwinds viral DNA and synthesizes primers for DNA replication, making it an attractive antiviral target. Although HP inhibitors pritelivir and amenamevir were identified through screening, their binding mechanisms remain unclear. Here, we report cryo-EM structures of HSV-1 HP bound to a forked DNA template alone, and in complex with pritelivir or amenamevir. The structures reveal a bilobed architecture highlighting helicase-primase coordinated action at the replication fork and providing a structural basis for HP inhibition by illustrating precisely how pritelivir and amenamevir block helicase activity. Data lays a solid foundation for the development of improved antiviral therapies.

biochemistry↗

bioRxiv2024

Nsp1 stalls DNA Polymerase alpha at DNA hairpins

The human primosome, a four-subunit complex of DNA primase and DNA polymerase alpha (Pol), plays a critical role in DNA replication by initiating RNA and DNA synthesis on both chromosome strands. A recent study has shown that a major virulence factor in the SARS-CoV-2 infection, Nsp1 (non-structural protein 1), forms a stable complex with Pol but does not affect the primosome activity. Here we show that Nsp1 inhibits DNA synthesis across inverted repeats prone to hairpin formation. Analysis of current structural data revealed the overlapping binding sites for Nsp1 and the winged helix-turn-helix domain of RPA (wHTH) on Pol, indicating a competition between them. Comparison of the inhibitory effect of Nsp1 and wHTH on DNA hairpin bypass by Pol showed an 8-fold lower IC50 value for Nsp1 (1 {micro}M). This study provides a valuable insight into the mechanism of inhibition of human DNA replication by Nsp1 during a SARS-CoV-2 infection.

biochemistry↗

bioRxiv2024

Human primosome requires replication protein A when copying DNA with inverted repeats

The human primosome, a four-subunit complex of primase and DNA polymerase alpha (Pol), initiates DNA synthesis on both chromosome strands by generating chimeric RNA-DNA primers for loading DNA polymerases delta and epsilon (Pol{varepsilon}). Replication protein A (RPA) tightly binds to single-stranded DNA strands, protecting them from nucleolytic digestion and unauthorized transactions. We report here that RPA plays a critical role for the human primosome during DNA synthesis across inverted repeats prone to hairpin formation. On other alternatively structured DNA, forming a G-quadruplex, RPA does not assist primosome. A stimulatory effect of RPA on DNA synthesis across hairpins was also observed for the catalytic domain of Pol but not of Pol{varepsilon}. The winged helix-turn-helix domain of RPA is essential for an efficient hairpin bypass and increases RPA-Pol cooperativity on the primed DNA template. Cryo-EM studies revealed that this domain is mainly responsible for the interaction between RPA and Pol. The flexible mode of RPA-Pol interaction during DNA synthesis implies the mechanism of template handover between them when the hairpin formation should be avoided. This work provides insight into a cooperative action of RPA and primosome on DNA, which is critical for DNA synthesis across inverted repeats. SIGNIFICANCEThis work revealed the critical role of RPA during DNA synthesis across inverted repeats by the human primosome. It was shown that a small winged helix-turn-helix domain of RPA is essential for the cooperative action of RPA and primosome, especially when copying DNA sequences prone to hairpin structure formation. Structural studies uncovered the mode of RPA and Pol integration, which assures an efficient DNA handover between them. This work provides a notable insight into the early steps of Okazaki fragments synthesis.

biochemistry↗

bioRxiv2023

The role of catalytic and regulatory domains of human PrimPol in DNA binding and synthesis

Human PrimPol possesses DNA primase and DNA polymerase activities and restarts stalled replication forks protecting cells against DNA damage in nuclei and mitochondria. The zinc-binding motif (ZnFn) of the C-terminal domain (CTD) of PrimPol is required for DNA primase activity but the mechanism is not clear. In this work, we biochemically demonstrate that PrimPol initiates de novo DNA synthesis in cis-orientation, when the N-terminal catalytic domain (NTD) and the CTD of one molecule take part in catalysis. The modeling studies revealed that PrimPol uses a similar mode of initiating NTP coordination as the human primase. The ZnFn motif residue Arg417 is required for binding the 5-triphosphate group that stabilizes the PrimPol complex with a DNA template-primer. We found that PrimPol is able to efficiently initiate DNA synthesis in the absence of the link between the two domains. The ability of the NTD alone to prime DNA synthesis and a regulatory role of the RPA-binding motif in the modulation of PrimPol binding to DNA are also demonstrated.

biochemistry↗

bioRxiv2022

DNA primase large subunit is an essential plant gene for geminiviruses, putatively priming viral ss-DNA replication

The family of Geminiviridae consists of more than 500 circular single-stranded (ss) DNA viral species that can infect numerous dicot and monocot plants. Geminiviruses replicate their genome in the nucleus of a plant cell, taking advantage of the hosts DNA replication machinery. For converting their DNA into double-stranded DNA, and subsequent replication, these viruses rely on host DNA polymerases. However, the priming of the very first step of this process, i.e. the conversion of incoming circular ssDNA into a dsDNA molecule, has remained elusive for almost 30 years. In this study, sequencing of melon (Cucumis melo) accession K18 carrying the Tomato leaf curl New Delhi virus (ToLCNDV) recessive resistance quantitative trait locus (QTL) in chromosome 11, and analyses of DNA sequence data from 100 melon genomes, showed a conservation of a shared mutation in the DNA Primase Large subunit (PRiL) of all accessions that exhibited resistance upon a challenge with ToLCNDV. Silencing of (native) Nicotiana benthamiana PriL and subsequent challenging with three different geminiviruses showed a severe reduction in titers of all three viruses, altogether emphasizing an important role of PRiL in geminiviral replication. A model is presented explaining the role of PriL during initiation of geminiviral DNA replication, i.e. as a regulatory subunit of primase that generates an RNA primer at the onset of DNA replication in analogy to DNA Primase-mediated initiation of DNA replication in all living organisms.

plant biology↗

bioRxiv2022

The iron-sulfur cluster is critical for DNA binding by human DNA polymerase ϵ

DNA polymerase {varepsilon} (Pol{varepsilon}) is a key enzyme for DNA replication in eukaryotes. It is attached to a helicase and performs DNA synthesis on the leading strand. Recently it was shown that the catalytic domain of yeast Pol{varepsilon} (Pol{varepsilon}CD) contains a [4Fe-4S] cluster located at the base of the processivity domain (P-domain) and coordinated by four conserved cysteines. In this work, we have shown that human Pol{varepsilon}CD (hPol{varepsilon}CD) expressed in bacterial cells also contains an iron-sulfur cluster. In comparison, recombinant hPol{varepsilon}CD produced in insect cells contains an eight-fold-lower level of iron. Interestingly, the iron content correlates with the level of DNA-binding molecules, which suggests an important role of the iron-sulfur cluster in hPol{varepsilon} interaction with DNA. Indeed, mutation of two conserved cysteines that coordinate the cluster abolished template:primer binding and, therefore, DNA polymerase and proofreading exonuclease activities. We propose that the cluster regulates the conformation of the P-domain, which, like a gatekeeper, controls access to a DNA-binding cleft for a template:primer. In addition, we performed kinetic and binding studies of hPol{varepsilon}CD. The binding studies demonstrated low affinity of hPol{varepsilon}CD to DNA and a strong effect of salt concentration on stability of the hPol{varepsilon}CD/DNA complex. Pre-steady-state kinetic studies have shown a maximal polymerization rate constant of 51.5 s-1 and a relatively low affinity to incoming dNTP with an apparent KD of 105 M. This work provides notable insight into the role of a [4Fe-4S] cluster in Pol{varepsilon} function.

biochemistry↗

bioRxiv2022

Insight into RNA-DNA primer length counting by human primosome

The human primosome, a four-subunit complex of primase and DNA polymerase alpha (Pol), synthesizes chimeric RNA-DNA primers for DNA polymerases delta and epsilon to initiate DNA replication on both chromosome strands. Despite recent structural insights into the action of its two catalytic centers, the mechanism of DNA synthesis termination is still unclear. Here we report results of functional and structural studies revealing how the human primosome counts RNA-DNA primer length and timely terminates DNA elongation. Using a single-turnover primer extension assay, we defined two factors that determine a mature primer length (~35-mer): 1) a tight interaction of the C-terminal domain of the DNA primase large subunit (p58C) with the primer 5-end, and 2) flexible tethering of p58C and the DNA polymerase alpha catalytic core domain (p180core) to the primosome platform domain by extended linkers. The obtained data allows us to conclude that p58C is a key regulator of all steps of RNA-DNA primer synthesis. The above-described findings provide a notable insight into the mechanism of DNA synthesis termination by a eukaryotic primosome, an important process for ensuring successful primer handover to replication DNA polymerases and for maintaining genome integrity.

biochemistry↗

bioRxiv2020

Replication protein A binds RNA and promotes R-loop formation

Replication protein A (RPA), a major eukaryotic ssDNA-binding protein, is essential for all metabolic processes that involve ssDNA including DNA replication, repair, and damage signaling. Surprisingly, we found here that RPA binds RNA in vitro with high affinity. Using native RIP method, we isolated RNA-RPA complexes from human cells. Furthermore, RPA promotes R-loop formation between RNA and homologous dsDNA. R-loops, the three-stranded nucleic acid structure consisting of an RNA-DNA hybrid and the displaced ssDNA strand, are common in human genome. R-loops may play an important role in transcription-coupled homologous recombination and DNA replication restart. We reconstituted the process of replication restart in vitro using RPA-generated R-loops and human DNA polymerases. These findings indicate that RPA may play a role in RNA metabolism and suggest a mechanism of genome maintenance that depends on RPA and RNA.

molecular biology↗