INVESTIGATING HOW THE ENDONUCLEASE MUTLα IS ACTIVATED AND SIGNALS IN DNA MISMATCH REPAIR

Abstract

In many DNA processes, action at a distance is required for signaling across long distances on DNA. These pathways, generally have an initiation site (site 1) that signals an event at a second location (site 2). Such a paradigm is found in processes such as transcription, replication, and DNA repair. To overcome long distances on DNA, proteins can utilize translocation, oligomerization, and DNA looping to bridge the distance between the initiating signal at site 1 and the site of action at site 2. The utilization of these mechanisms for action at a distance is crucial in eukaryotic mismatch repair. In this pathway, MutS homologs scan DNA and recognize mis-paired bases. The MutS protein then recruits the endonuclease MutLα, which nicks the nascent strand of DNA containing a mis-incorporated DNA base. The MutLα-generated nick leads to downstream mis-pair removal through excision by an exonuclease or strand displacement activities of a DNA polymerase working together with a flap endonuclease. Although, previous models have suggested that MutL homolog endonucleases can form oligomeric complexes on DNA, the role of a MutLα oligomeric complex and how it might facilitate action at a distance has been unclear. Here, I present evidence that the mismatch repair MutLα endonuclease is activated by DNA-DNA associations, and it can use this activity to overcome DNA torsional barriers. Using DNA ligation and pull-down experiments, I determined that a MutLα oligomer associates two DNA duplexes and that this activity can stimulate MutLα’s endonuclease function. I also show evidence that MutLα enhances a topoisomerase without nicking the DNA itself. These behaviors of MutLα could localize nicking on DNA near a mismatch and help overcome barriers that could inhibit additional repair proteins from activating MutLα and facilitating efficient DNA repair. The endonuclease activity of MutLα is critical for efficient mismatch repair, but in addition to this activity, MutLα is also an ATPase, although the crosstalk between the two enzymatic functions has been largely unexplored. It has been shown previously that the ATPase activity of MutLα allows the protein to undergo conformational changes and in vivo is necessary for efficient mismatch repair. Mechanistically, how this activity supports MutLα’s functions in the mismatch repair pathway remains unclear. Using DNA binding and photo-crosslinking experiments, I provide evidence that MutLα recognizes and localizes itself to a nick. Additionally, through DNA protection assays and photo-crosslinking I provide evidence of a signaling mechanism initiated at the nick for a MutLα oligomer to undergo its ATP cycle. These data provide insight into how MutLα uses ATP to signal events for mismatch removal. These data also provide a mechanistic explanation for how MutL proteins interact with DNA during mismatch repair and send signals for additional repair processes after the protein nicks DNA that help explain new models for action at a distance.