Structure-function analysis of SLX4 and its scaffolded nucleases

Thesis

The ever-expanding utility of cryoelectron microscopy (cryo-EM) has enabled resolving architectures of protein complexes that were once considered beyond reach. However, DNA damage-associated protein complexes pose unique challenges due to their pronounced flexibility and intrinsic disorder, which render them poorly defined even by advanced in silico machine-learning-based structural predictions. Such complexes denote high- priority but structurally elusive targets, necessitating careful purification and experimental structure determination.

One such complex is the SLX4-scaffolded nuclease complex (SMX) comprising the DNA nucleases XPF-ERCC1, MUS81-EME1 and SLX1. This complex is profoundly implicated in cancer, ageing, and Fanconi anaemia (FA) - diseases intricately linked to DNA damage and repair mechanisms. SLX4 serves as a platform for recruiting these nucleases and coordinates the resolution of diverse DNA lesions, particularly interstrand crosslinks (ICLs), which are among the most cytotoxic forms of DNA damage. The structural characterisation of the SMX complex remains a critical and ambitious objective of this project. Furthermore, endogenous causative agents generating ICLs in cells remain an area of active investigation.

This work contributes to the growing evidence implicating formaldehyde as a key endogenous source of ICLs, supporting its intrinsic connection to aldehyde toxicity and the Fanconi anaemia pathway. This study further proposes that the accumulation of formaldehyde during epigenetic reprogramming may represent a significant source of ICLs, potentially contributing to the differentiation defects observed in the blood lineages of patients with FA. In parallel, CRISPR-Cas9-targeted disruption of SLX1 and XPF was undertaken to determine biological roles and genetic interactions underlying their loss. Moreover, significant efforts were made toward the homogeneous purification of SMX, employing multi-pronged expression systems to address obstacles in achieving stability and functionality. Given challenges associated with purifying the entire complex, functional studies on sub-complexes and investigations into novel interactions of SLX4 provide valuable insights into the post-translational modifications regulating SLX4’s association with MUS81-EME1 and other DNA repair factors. The ultimate goal of these efforts is to obtain high-quality SMX suitable for structural characterisation by cryo-EM, advancing understanding of its molecular architecture and biological roles.

Authors: Ratnaweera, M

• DOI: 10.5287/ora-gavgboevk
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: interstrand crosslinks; DNA damage repair; Fanconi anaemia; SLX4; Cancer
• Subjects: DNA damage; DNA repair; Fanconi's anemia