Biology subjects

Lobrich, M.

Publications and source records attributed to Lobrich, M..

3 recordsLinked to original sources

bioRxiv2026

Structures and molecular mechanisms of RAD54B in modulating homologous recombination

Genome stability is essential for cellular viability yet constantly threatened by endogenous and exogenous DNA-damaging agents. Among these, DNA double-strand breaks (DSBs) are particularly harmful and in S/G2 phases are faithfully repaired through homologous recombination (HR), a high-fidelity pathway utilising homologous sequences in sister chromatin. The RAD51 recombinase forms nucleoprotein filaments on single-stranded DNA (ssDNA) to mediate homology search, strand invasion and subsequent D-loop formation that leads to DNA synthesis and repair. The efficiency of HR depends on precise regulation of RAD51 filament dynamics by accessory factors, including RAD54 and RAD54B, which belong to the SWI2/SNF2-family DNA translocases. While RAD54 is well-characterized, RAD54Bs molecular functions remain poorly understood. Here, we define RAD54Bs role in HR using cryo-electron microscopy, mutagenesis, biochemical and cellular assays. We show that RAD54B stabilizes RAD51-DNA filaments, inhibits RAD51 ATPase activity, and promotes strand invasion, D-loop formation and strand exchange. The N-terminal domain (NTD) alone supports filament stabilization and strand exchange, while the C-terminal ATPase domain is required for D-loop formation. Structural and biochemical analyses reveal three RAD51-interacting sites within the NTD and a unique domain ({beta}-domain) that bridges RAD51 protomers and contacts donor dsDNA. This {beta}-domain also regulates RAD54Bs ATPase activity and higher-order oligomer organization on dsDNA. Cellular assays reveal that the NTD RAD51-interacting sites as well as the {beta}-domain are required for repairing camptothecin-induced DSBs by HR in human cells. Our findings uncover a modular architecture and mechanistic framework for RAD54B function in HR, highlighting its critical role in genome maintenance. HighlightsO_LIcryoEM structure of RAD54B in complex with RAD51-DNA complex C_LIO_LIRAD54B uses three sites to interact with RAD51, including a previously unrecognised {beta}-domain that bridges distal RAD51 protomers. C_LIO_LIThe {beta}-domain plays multiple crucial roles including regulating filament stability, RAD54B ATPase activity and RAD54B higher order assembly on DNA. C_LIO_LIRAD54B employs a modular mechanism, with the N-terminal region engaing and stabilising RAD51 filaments, capturing of the homologous strands, whereas the ATPase motor domainrequired for homology search and strand invasion. C_LIO_LIRAD54B N-terminus and {beta}-domain are essential for HR-mediated repair of camptothecin-induced breaks in human cells. C_LI

biochemistry↗

bioRxiv2025

Repair of DNA double-strand breaks after low radiation doses in childhood cancer survivors and matched cancer-free individuals

DNA double-strand breaks (DSBs) which arise in G1- or G0-phase normal human cells are repaired by non-homologous end-joining (NHEJ), a pathway which is important for cell survival but can cause mutations at the break sites. DSB repair by NHEJ is very efficient at high damage levels of 1 or more DSBs per cell, much less efficient at lower damage levels and almost absent if only [~]0.05 DSBs per cell are induced. Repair at this low DSB level can be induced if cells are pre-treated prior to DSB induction with low concentrations of hydrogen peroxide, suggesting that the intracellular radical level modulates the efficiency of DSB repair at low damage levels. Here, we have investigated if the inefficiency of repair at low DSB levels contributes to the carcinogenic potential of DSBs or, counterintuitively, may serve as a mechanism to limit cancer development. We have analyzed the repair of high and low levels of radiation-induced DSBs in primary fibroblasts from 136 childhood cancer survivors, half of whom developed a second independent tumor later in life, and compared it to the response of primary fibroblasts from 68 individually matched cancer-free individuals. Although childhood cancer survivors and cancer-free individuals repaired DSBs at high damage levels equally efficiently, their response to low DSB levels differed drastically. While repair in cancer-free individuals was nearly absent at a level of [~]0.05 DSBs per cell, childhood cancer survivors repaired DSBs at this low damage level as efficiently as after high damage levels. These results suggest that most of the cancer survivors analyzed here carry a genetic predisposition that affects their response to low levels of DSBs. They also support the idea that the absence of repair observed in cancer-free individuals represents a mechanism limiting cancer formation.

cell biology↗

bioRxiv2025

Nek family members regulate Rad54 during homologous recombination in developing mice

Homologous recombination (HR) represents an important pathway for repairing DNA double-strand breaks (DSBs) but HR factors, including RAD51, also serve to protect and restart stalled replication forks. RAD54 functions during DSB repair where it removes RAD51 from duplex DNA including heteroduplex DNA which is formed during D-loop formation. This allows subsequent DNA repair synthesis and completion of the HR process. We have previously suggested that RAD54s activity is regulated by never-in-mitosis-gene A (NIMA) related kinase 1 (NEK1) in a cell cycle-specific manner to promote RAD51 removal and HR in G2 phase without interfering with RAD51s fork stabilization role during S phase. Here, we establish that Nek1 regulates the phosphorylation of Rad54 at Ser572 (S572) to promote HR in vivo in adult mice and in vitro in fibroblasts derived from such mice. In contrast, embryonic mice and fibroblasts derived from them do not require Nek1 for HR. We further show that HR requires Rad54 phosphorylation at S572 both in embryonic and adult fibroblasts and that this is mediated in embryonic fibroblasts by Nek3 and Nek5 instead of Nek1. Thus, our work identifies a developmental change in the regulation of HR and uncovers two new factors involved in this process.

cell biology↗