How do Smc5/6 interactions with DNA coordinate replication and recombination?

AE1: DNA replication AE2: DNA repair double strand breaks/ Smc5/6 AE3: double-strand break repair mechanism AE4: SMC/cohesin-DNA protein structure

Structural Maintenance of Chromosomes (SMC) complexes (condensin, cohesin and Smc5/6) play critical roles in orchestrating chromosome dynamics throughout the cell cycle and are essential for the maintenance of genomic integrity. Smc5/6 regulates homologous recombination and replication restart activities, both key to genome stability.
We have shown that the hinge-domain of Smc5/6 binds preferentially to single-strand DNA and that the NSMCE1/3/4 subcomplex binds preferentially to double-strand DNA (dsDNA). We propose that these two DNA-binding activities, separated by ~50nm, work together to control, regulate and ultimately resolve complex DNA structures such as homologous recombination and replication intermediates.
To investigate and test our hypothesis, we will co-crystallise the Smc5/6-hinge and NSMCE1/3/4 accessory complex with DNA, thus determining the molecular details underpinning each interaction type. For both complexes, we will also look at any concomitant changes in conformation driven by DNA-binding, using SAXS and other biophysical methods, such as FRET. With the structural information obtained, we will create and test defined mutations (in vitro and vivo) for effects on biological function.
We will isolate sufficient Smc5/6 holo-complex to enable single-particle electron microscopy and/or AFM-based approaches, examining the structure and conformational changes, in the presence and absence of bound DNA. We will determine if the holo-complex binds preferentially to biologically-relevant DNA structures, such as Holliday junctions, D-loops and fork-like structures, and whether this can occur in the presence of RPA (replication-protein A), and if DNA-interaction brings the head and hinge regions together.
These studies will provide insight into the structure, biochemistry and in vivo function of this important complex and lead to an understanding of how Smc5/6 coordinates replication and recombination and its role in maintaining human health.

This project aims to elucidate how Smc5/6 interactions with DNA coordinate replication and recombination and to understand how such interactions affect Smc5/6 conformation and function. Thus, the project will benefit the Smc5/6 and SMC research communities. It will inform the wider DNA repair and recombination fields and has important implications for our understanding of related pathways in higher eukaryotes.

It will advance our understanding of the role Smc5/6 in maintaining human health. Smc5/6 DNA binding underpins its role in preventing lung disease, maintaining genetic stability and likely as a restriction factor for Hepatitis B infection. Therefore, the proposal will have significant impact for research progress in these areas, as well as contributing to the understanding of diseases related to DNA repair defects, such as cancer.

The potential to therapeutically modulate Smc5/6 function, for example to exploit synthetic interactions in cancer, will be enhanced by a detailed molecular understanding. It will also impact on treatments for Hepatitis B and potentially other viral infections. This work will thus contribute to improvements in the quality of life.

The proposal will also provide training for the next generation of scientists in cutting edge techniques and transferable skills, impacting on the creation of a skilled workforce.

Lastly, by developing collaborations between researchers in different disciplines and Schools it will impact on the development of inter-disciplinary research.