Biology subjects

Baranovskiy, A. G.

Publications and source records attributed to Baranovskiy, A. G..

6 recordsLinked to original sources

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↗

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

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↗