Structural and molecular mechanisms of the 55LCC ATPase complex and its function in DNA replication

Replication of human DNA is a cellular process required for the faithful transfer of genetic information from parents to progeny. This is driven by the timely assembly and disassembly on and off DNA of a complex molecular machine, known as the replisome. Cells have evolved sophisticated protein quality control mechanisms that ensure removal of replisome factors from chromatin, including protein unfolding and proteolytic turnover.

Regulation of this cellular event is of central importance to ensure correct DNA replication and genome stability. The archetypical AAA+ unfoldase VCP/p97 is a major player in this process, and fascinating mechanistic insights of VCP/p97-dependent protein unfolding and degradation have been discovered over the past decade. However, it is not clear if VCP/p97 is the only AAA+ protein involved in regulating DNA replication and genome stability, or if other ATPase-driven macromolecular motors are also involved.

We have recently identified the AAA+ ATPases SPATA5 and SPATA5L1 as key players in the proteolytic turnover of replisome factors during S-phase. We discovered that SPATA5-SPATA5L1, in complex with the heterodimeric partners C1orf109-CINP, form an enzymatic assembly (referred to as 55LCC) that interacts with components of the replication machinery and mediates their ATPase-dependent unfolding.

Our work provides critical functional insights on a new macromolecular complex, and describes a new paradigm for replisome turnover on chromatin. Despite these considerable advances, a precise molecular and mechanistic understanding of how SPATA5 and SPATA5L1 modulate and contribute to 55LCC ATPase activity, substrate engagement and unfolding is still lacking.

Throughout this Discovery Fellowship I aim to understand the molecular and mechanistic underpinnings of how 55LCC complex assembly directs substrate recruitment and regulates 55LCC enzymatic function. I will address three main questions: 1) How do SPATA5 and SPATA5L1 contribute to 55LCC enzymatic activities? I will use biochemical approaches to generate a complete mechanistic understanding of the specific contributions SPATA5 and SPATA5L1 provide to 55LCC ATPase and unfoldase function. 2) What are the structural determinants and conformational dynamics of SPATA5 and 55LCC?

I will combine structural, biophysical and computational methods to gain insights into the determinants underpinning SPATA5 and 55LCC assemblies, and clarify the contributions SPATA5 and SPATA5L1 provide to 55LCC structural integrity and function. In doing this I will also obtain a deeper understanding of the conformational dynamics and activity-driven changes sampled by SPATA5 and 55LCC in different nucleotide-bound states. 3) How does 55LCC associate with its substrates?

I will use biochemical and biophysical approaches to quantitatively characterise 55LCC interaction with its biological substrates, and generate critical mechanistic information on how substrate recognition and processing is regulated by 55LCC.

The outlined proposal will transform our understanding on how this new protein complex functions as a macromolecular machine, and generate new molecular and mechanistic insights into 55LCC complex assembly, enzymatic activities and substrate recruitment. The outcomes generated throughout this Fellowship will also provide a strong platform for follow-up studies, including structural biology analyses of 55LCC:substrate assemblies as well as ligand/drug discovery projects within academic settings or by establishing connections with future industry partners.