Procedure no. DSMBBC/2027/08: DNA twist and topology in target search by DNA-binding proteins in Katowice

Institute of Biochemistry and Biophysics Polish Academy of Sciences

Organisation/Company Institute of Biochemistry and Biophysics Polish Academy of Sciences Research Field Biological sciences Researcher Profile First Stage Researcher (R1) Positions PhD Positions Application Deadline 30 Nov 2026 - 23:59 (Europe/Warsaw) Country Poland Type of Contract Temporary Job Status Full-time Hours Per Week 40 Offer Starting Date 1 Mar 2027 Is the job funded through the EU Research Framework Programme? Not funded by a EU programme Is the Job related to staff position within a Research Infrastructure? No

Offer Description Background. Proteins that target specific genomic sites - repair enzymes, recombinases, transcription factors - must locate them among millions of near-identical sequences. It is well established that they do this by facilitated diffusion, alternating between 3D diffusion in solution and 1D sliding along DNA. Most of these observations were made in vitro on topologically relaxed DNA. In cells, however, DNA is kept slightly underwound, and transcription, replication and loop extrusion constantly add local twist. Live-cell imaging, in turn, preserves this native topological state but cannot control or measure it locally. Recent single-molecule work shows the emerging supercoiling matters - for example underwinding of the DNA makes Cas9 bind and cut at multiple off-target sites (Newton et al., 2023), and RecA filaments only engage with homologous sequences when that target is negatively supercoiled (Tišma et al., 2026). Methodological challenge. Existing methods to supercoil DNA in single-molecule imaging rely mainly on intercalators, which alter twist and helix stability. The main alternative avoids dyes but requires pulling the DNA far beyond its normal length first, which damages the duplex. In this project, we will establish a new method for generating defined negative supercoiling in optical tweezers, free of intercalators and without mechanical overstretching, reaching physiological supercoiling densities. This method will be independently validated on magnetic tweezers, where twist can be applied directly. Research question. Does underwinding change how a protein moves along DNA, or how it recognises its target site? Model system. The project will start with Fpg, a bacterial glycosylase that finds and removes 8-oxoguanine, the most common oxidative lesion in DNA. It is a well characterised system: its sliding along DNA has been described in detail, but mainly on relaxed DNA, and not under controlled torsion. Fpg will give a direct mechanical readout - after removing the damaged base it cuts the DNA backbone, and a single cut releases all the torsional strain at once. In parallel, we will develop a strategy to purify a fluorescently labelled protein to monitor its sliding on torsionally constrained DNA using fluorescence-coupled optical tweezers. Once established, the same assay can be applied to