Wrapping, threading and cutting: a single-molecule perspective on DNA processing enzymes

The integrity of the genome is being continuously challenged by a variety of damaging agents including UV light and chemical compounds that cause mutations with severe consequences for human health. To maintain intact the genetic code, all organisms devote a considerable amount of energy to protect, and when that fails, to repair damaged DNA.
In the first part of this talk we will explore, at single-molecule level, the function of two DNA repair protein complexes conserved across all domains of life that act as molecular scissors cutting branched DNA structures. Flap endonuclease 1 (Fen1) and Xeroderma Pigmentosum Group F (XPF) are two DNA repair enzymes that process 5’ and 3’ DNA flaps, respectively. In humans, these flap structures are generated 5 million times per cell cycle during lagging strand replication and failure to eliminate them would compromise cell viability. For instance, Fen1 mutations are known to cause predisposition to cancer and mutations in the XPF gene cause Xeroderma Pigmentosum (XP), which is characterized by extreme sensitivity to UV light and a high frequency of skin cancer. Both enzymes require Proliferating Cell Nuclear Antigen (PCNA) to efficiently process the flap structure but we will show that PCNA activates each enzyme following a very different mechanism.
In the second part we will focus on understanding how a monomeric single-strand DNA binding protein (SSB) wraps the single-strand DNA sequence to occlude the nucleobases and protect the genomic information. We will demonstrate that this SSB protein operates using a delicate balance between maximal occupancy of the ssDNA to ensure efficient protection and a highly dynamic interaction with ssDNA to allow access to ssDNA during vital cellular processes.