Biology subjects

Korzhnev, D.

Publications and source records attributed to Korzhnev, D..

3 recordsLinked to original sources

bioRxiv2026

Mutation of charged inner pore residues reduce E. coli β clamp residency and increase sliding rates on DNA

Sliding clamp proteins play central roles in DNA metabolism, including replication and repair. The ring-shaped E. coli beta clamp accommodates double-stranded DNA and serves as a platform for proteins involved in multiple DNA transactions. The inner pore of the beta clamp harbors a series of positively charged and polar residues that can bind to the negatively charged backbone of the DNA. These residues are arrayed so that they do not align with the charged phosphates of the DNA backbone. It is hypothesized that this arrangement of these residues provides for the movement of the clamp on DNA as it alternates which residues are bound to the DNA backbone. In this work, we mutated specific charged and polar residues that project into the inner pore of the beta clamp. The beta clamp variants are dimers and have similar thermal stability and in general a similar ability to complement a temperature sensitive strain for growth. One exception was beta-Q149A, which appeared as higher-order species on a native gel although its hydrogen-deuterium exchange pattern measured by mass spectrometry was overall similar to WT beta. These variants all had decreased binding to DNA after loading. Optical tweezers experiments were used to monitor loading on single DNA molecules and measure the rate of beta clamp sliding on DNA. Consistent with the hypothesized role of positively charged residues in the beta inner pore, mutation of one residue resulted in a faster rate of sliding on DNA.

biochemistry↗

bioRxiv2026

A RAD18 SAP domain PIP motif enables PCNA mono-ubiquitination and USP1-BRCA1 synthetic lethality

The proliferating cell nuclear antigen (PCNA) sliding clamp is mono-ubiquitinated by RAD6-RAD18 in response to DNA damage, initiating the DNA damage tolerance pathway of translesion synthesis. The molecular basis by which RAD18 engages PCNA has, however, remained incompletely defined. Mono-ubiquitinated PCNA is subsequently poly-ubiquitinated with K48-linked chains that target PCNA for degradation. Ubiquitin-specific protease 1 (USP1) reverses PCNA mono- and poly-ubiquitination; accordingly, inhibiting USP1 causes the accumulation of mono-ubiquitinated PCNA at replication forks and a reduction in total PCNA levels. USP1 inhibitors promote the accumulation of ssDNA gaps (ssGAPs) in newly replicated DNA and are synthetic lethality in BRCA1-deficient cells. Here, we combine computational and structural approaches to identify and characterize a PCNA-interacting peptide (PIP) motif in RAD18. This PIP motif is required for RAD18-dependent DNA damage-induced PCNA ubiquitination and PCNA turnover. Mutation of the RAD18-PCNA interface reduces ssGAP accumulation and USP1 inhibitor sensitivity in BRCA1-deficient cells. Furthermore, cells adapted to prolonged USP1 inhibition exhibit reduced RAD18 levels, suggesting that deregulation of RAD18 contributes to a biologically relevant drug resistance mechanism. This resistance could be overcome by inhibiting the Ataxia telangiectasia and Rad3-related (ATR) kinase. Together, these findings define a molecular interface required for RAD18-dependent PCNA mono-ubiquitination and identify it as a key determinant of USP1-BRCA1 synthetic lethality.

biochemistry↗

bioRxiv2025

REV1 inhibition enhances trinucleotide repeat mutagenesis

Trinucleotide repeat (TNR) instability has been implicated in the pathogenesis of numerous neurodegenerative disorders. Because TNR instability causes mutagenesis of the underlying gene, we refer to the repeat instability phenomenon as TNR mutagenesis in this study. While germline expansions destabilize TNR to cause disease anticipation, somatic cell TNR instability drives earlier onset of symptoms and further disease progression. However, the drivers behind these repeat length changes remain unclear. Current models suggest that DNA replication slippage events and the action of genome instability pathways, such as DNA repair, cause TNR mutagenesis. Whether mutagenic polymerases from the translesion synthesis (TLS) pathway result in TNR instability is unclear. TLS polymerases are best at bypassing difficult-to-replicate DNA regions due to bulky lesions or gaps in DNA. While some effects of TLS polymerases on TNR instability have been explored in lower organisms, evidence in human cells is lacking. Using a quantitative GFP reporter with expanded CAG repeats, we show that inhibition of the TLS polymerase REV1 by its inhibitor, JH-RE-06, or siRNA knockdown increases TNR instability and the underlying mutability. These results suggest that REV1 protects Trinucleotide repeat length mutagenesis through potential continuous DNA synthesis when replicative polymerases stall ahead of repeat secondary structures. Collectively, we present evidence of the role of the TLS pathway in TNR instability, with potential implications for understanding mutability mechanisms, disease biology, and therapeutic targeting.

genetics↗