Biology subjects

Kutuzov, M. M.

Publications and source records attributed to Kutuzov, M. M..

5 recordsLinked to original sources

bioRxiv2026

More than just a passive brick in the wall: the nucleosome facilitates DNA polymerase β activity in linker DNA and its PARP-dependent regulation in the BER pathway choice

DNA polymerase {beta} (Pol{beta}) is a central player of base excision repair (BER), performing gap-filling synthesis on damaged DNA. While nucleosome core particles (NCPs) are known to impede activity of BER enzymes, the regulation of this process in linker DNA adjacent to nucleosomes remains unclear. Here we demonstrate an unexpected stimulation of Pol{beta}-catalyzed gap-filling and strand-displacement synthesis in linker DNA by the adjacent NCP. Notably, the nucleosomal context reinforces the regulatory modulation of Pol{beta} activity by PARP1/PARP2 and FEN1. While linker histone H1 restricts strand-displacement synthesis at the nucleosome entry/exit site, PARP1 and PARP2 modulate Pol{beta} function through competitive binding to DNA gaps or nicks and via poly(ADP-ribosyl)ation (PARylation). At the same time, PARPs binding differentially regulates BER sub-pathway choice, and PARylation alleviates H1-mediated inhibition. These findings reveal a multi-layered regulatory system wherein the nucleosome acts as a dynamic platform coordinating Pol{beta} activity and its interplay with chromatin-associated factors, influencing the balance between short- and long-patch BER. The research advances understanding of chromatin-mediated control of BER DNA repair synthesis and the functional specialization of PARP1 and PARP2 in maintaining genome stability. HighlightsThe nucleosome core particle acts not only as a barrier but also as a stimulator of Pol{beta}-mediated DNA repair synthesis in adjacent linker DNA. The nucleosome acts as an allosteric platform that enhances the regulatory functions of chromatin-associated factors (PARP1, PARP2, H1) in the linker DNA repair synthesis. PARP1 suppresses overall Pol{beta} synthesis, while PARP2 specifically inhibits strand displacement, thereby gating the choice between short- and long-patch BER pathways.

molecular biology↗

bioRxiv2026

Poly(ADP-ribose)polymerase1 facilitates the nucleosome disassembly

Being the basic building blocks of chromatin, nucleosomes and their stability determine the genome accessibility for different DNA-dependent proteins. This characteristic is labile under all cell-life processes. One of the abundant DNA-binding proteins, which is important for genome compaction, is poly(ADP-ribose)polymerase1 (PARP1). Despite the extensive experimental data on the chromatin compaction regulation under ADP-ribosylation, the details of the interplay of nucleosome with PARP1 in the absence of protein activation remain unclear. In this study, we analyzed the changes in the nucleosome wrapping upon PARP1 interaction using a single-molecule approach -- optical tweezers. We demonstrate that PARP1 binding leads to weakening of the contacts that support the nucleosome core.

molecular biology↗

bioRxiv2025

Interplay between human ribosomal proteins, PARP1, PARP2, HPF1 and histones

ADP-ribosyl-transferases (ADP-ribose polymerases) PARP1 and PARP2 are critical players in DNA damage response in the nucleus. Being activated by a genotoxic stress, these enzymes utilize NAD+ to attach ADP-ribose chains to wide variety of proteins; ribosomal proteins (RPs) have been identified among the major targets of the modification in different cell lines. However, little remained known concerning the peculiarities of the reaction of RPs ADP-ribosylation itself. Here, we study ADP-ribosylation of human RPs within the large (60S) and small (40S) ribosomal subunits and those isolated from the subunits, with PARP1 and PARP2 in vitro using radioactively labeled NAD+. We fail to detect the modification of ribosome-bound RPs but observed ADP-ribosylation of certain ribosome-free RPs when we use total protein isolated from the subunits. RPs from the 60S subunit were globally more modified than those from the 40S subunit, and ADP-ribosylation of several 60S RPs (but not 40S) was considerably enhanced in the presence of histone PARylation factor 1 (HPF1). With all kind RPs, HPF1 switches the modification preferentially to their serine/tyrosine residues. Major targets of the 60S RPs ADP-ribosylation were identified as RPL4 (uL4), RPL6 (eL6) and RPL13A/RPL15 (uL13/eL15). The modification levels of particular RPs differently depend on the concentration of total RP; the most selective HPF1-dependent ADP-ribosylation occurs in RPL6 (eL6). When present simultaneously with histones, RPs win linker histone H1 in the competition for both PARPs; in contrast, core histones strongly compete with RPs for ADP-ribosylation. Possible functional assignments of ADP-ribosylation of RPs are discussed. Bullet points- Free human ribosomal proteins are PARylated by PARP1 and PARP2; - PARylation of ribosomal 60S proteins but not 40S ones is mostly HPF1-dependent; - RPL4, RPL6 and RPL13A/RPL15 are the major targets of PARylation among 60S RPs; - Linker histone H1 is a poor competitor to ribosomal proteins for PARPs; - Core histones strongly competes with ribosomal proteins for PARPs.

molecular biology↗

bioRxiv2025

HPF1 REGULATES THE FORMATION OF FUS-DEPENDENT COMPARTMENTS BY PARP1 AND PARP2 ACTIVATION ON DAMAGED DNA

FUS participates in the formation of biomolecular condensates associated with PARP1-dependent synthesis of poly(ADP-ribose) (PAR). HPF1 regulates auto- and hetero-PARylation activities of PARP1 and PARP2 and may influence the formation of FUS compartments during PARP1 or PARP2 auto-PARylation. In this study, we used atomic force microscopy in combination with biochemical assay to investigate the formation of FUS compartments under activation of PARP1 and PARP2, when HPF1 modulates their activity. Similar to PARP1, FUS and PARylated PARP2 form DNA-rich compartments, indicating that PARP2 PARylation is sufficient for the formation of such compartments. The excess of HPF1 over PARP1 diminishes PARP1 activity and reduces the size of DNA-rich compartments. However, an excess of HPF1 over PARP2 does not significantly affect PARP2 activity and the size of compartments. Furthermore, HPF1 stimulates hetero-PARylation of FUS; this modification is stronger with PARP2 than with PARP1. HPF1-dependent intensive PARylation of FUS catalyzed by PARP1 or PARP2 impairs the assembly of DNA-rich compartment. These data provide a basis for investigating the effect of HPF1 on the formation of PAR-dependent condensates involving RNA-binding proteins like FUS, which interact effectively with PAR and show the ability to be targets of PARylation to regulate condensate formation at DNA damage sites. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/673448v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1fb3dadorg.highwire.dtl.DTLVardef@18e3b4forg.highwire.dtl.DTLVardef@d7e22forg.highwire.dtl.DTLVardef@14293af_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Cas9 is mostly orthogonal to human systems of DNA break sensing and repair

CRISPR/Cas9 system is [a] powerful gene editing tool based on the RNA-guided cleavage of target DNA. The Cas9 activity can be modulated by proteins involved in DNA damage signalling and repair due to their interaction with double- and single-strand breaks (DSB and SSB, respectively) generated by wild-type Cas9 or Cas9 nickases. Here we address the interplay between Streptococcus pyogenes Cas9 and key DNA repair factors, including poly(ADP-ribose) polymerase 1 (SSB/DSB sensor), its closest homolog poly(ADP-ribose) polymerase 2, Ku antigen (DSB sensor), DNA ligase I (SSB sensor), replication protein A (DNA duplex destabilizer), and Y-box binding protein 1 (RNA/DNA binding protein). None of those significantly affected Cas9 activity, while Cas9 efficiently shielded DSBs and SSBs from their sensors. Poly(ADP-ribosyl)ation of Cas9 detected for poly(ADP-ribose) polymerase 2 had no apparent effect on the activity. In cellulo, Cas9-dependent gene editing was independent of poly(ADP-ribose) polymerase 1. Thus, Cas9 can be regarded as an enzyme mostly orthogonal to the natural regulation of human systems of DNA break sensing and repair.

biochemistry↗