title: Investigating the importance of RAD54 and NEK1 in homologous recombination within the developing mouse
Creators: name: Thomas, Holly
Publisher: name: UNSPECIFIED
Year: 2022

description: Mammalian development begins in the zygote whereby regulated mass cell expansion takes place to create a living organism. However, efficient cell expansion relies on multiple mechanisms including, the maintenance of genomic integrity. DNA damage can originate from endogenous and exogenous sources and cells rely on the use of specific repair pathways to repair a genotoxic insult. Types of damage include single-strand breaks, double-strand breaks (DSBs), base mismatches and bulky adducts. Of these DSBs represent one of the most dangerous types of lesions a cell can encounter. If a DSB is left unrepaired or is mis-repaired, then genomic stability and cell viability are at risk. Two primary mechanisms are available to repair DSBs in eukaryotic cells and the choice of pathway is dependent on various factors including the cell cycle phase. The first is, non-homologous end joining (NHEJ) which operates throughout the cell cycle and in G0. The second, homologous recombination (HR), provides a high-fidelity mechanism of error-free repair and is only available during S and G2 phases, due to the need for a second homologous sequence, typically the sister chromatid, for DNA synthesis. During later steps of HR, after a process called resection, two strands of single-stranded DNA (one on either break site) are coated with a protein called RAD51. This enables a homology search to find the homologous sequence of DNA on the sister chromatid. RAD51 is removed by the repair protein RAD54 so DNA synthesis can proceed, leading to the completion of repair. Over the past 30 years, an expansion of studies has been published concerning the mechanisms surrounding HR. One important publication defines the regulation of RAD54, in an in vitro human model system. Spies et al. (2016) describe how the kinase NEK1 phosphorylates RAD54 specifically in G2 phase which activates RAD54 to remove RAD51 from ssDNA. Via the use of phospho-mimic and phospho-mutant RAD54 cell systems, Spies et al. found that the premature phosphorylation of RAD54 in S phase results in early removal of RAD51 from the ssDNA, resulting in fork degradation and a repair defect, therefore highlighting the importance in the timing of the regulation. But, is NEK1 responsible for regulating RAD54 in vivo? This study investigates the role of NEK1 in vivo, using knockout mouse strains of Nek1 and Rad54. As HR is extremely important and vastly utilised during early development, experiments were conducted in embryonic, neonatal and juvenile mice. A novel in vivo immunofluorescence (IF) staining assay identifies HR repair alongside cell cycle progression and assesses DSBs undergoing HR after ionising radiation (IR). Specifically, organs of the central nervous system (CNS) and the small intestine were used. A comparison of the DNA damage response therefore can be ascertained between tissues and developmental stages following IR. Loss of RAD54 leads to an HR repair defect after IR in all mouse tissues analysed. However, the scale of the defect varies in a tissue-dependent manner. Quantitative analysis in the unirradiated Rad54-/- mice showed a potential role of RAD54 in replication but in a tissue and developmental-specific manner. Interestingly the role of NEK1 differs during development with an HR repair defect only being present in the juvenile developmental stage. IF imaging confirms this change in the role of NEK1 throughout development with NEK1 localising in the cytoplasm during early development but then localises to the nucleus by the juvenile developmental stage. Nek1-/- mice also develop a later onset phenotype including growth retardation which is only apparent between the neonatal and juvenile stages. Taken together, these results would indicate a differential role for NEK1 during development.