DNA replication and mismatch repair system coupling using single molecule technology.

LUISINA DE TULLIO; ERIC C. GREENE

Vieques

Congreso; Pew Biomedical 2015 Annual Meeting.; 2015

The enzymatic synthesis of DNA is a process performed with high fidelity. Nevertheless, when DNA polymerase makes a mistake, there are numerous cellular mechanisms that prevent replication errors and genomic instability. Among these mechanisms, the postreplicative mismatch repair (MMR) system contributes to the conservation of DNA integrity by correcting base-base mismatches, and small insertion/deletion loops produced during DNA replication. The Proliferating Cell Nuclear Antigen (PCNA) has been postulated as the link between the replication machinery and the MMR system, because it is required for DNA polymerase processivity, and a PCNA binding motif has been characterized in MutSα. However, the precise relation of these two mechanisms at a mechanistic and molecular level has yet to be elucidated. Using DNA curtains it is possible to visualize in real-time, at a single molecule level, the tracking of proteins along linear DNA. This is achieved using bright photostable fluorescent molecules like quantum dots for protein labeling, and a total internal reflection fluorescence microscopy (TIRFM) for imaging detection1,2. In this project we will attempt to study the spatial-temporal coupling between DNA replication, and the MMR system of Saccaromyces cerevisiae. For this, we will adapt the in vitro reconstitution of the S. cerevisiae replication machinery previously performed in Dr. Stephen Bell?s laboratory (MIT)3 for use in the DNA curtain assays. The strategy undertaken will involve the reconstitution of replisomes on DNA curtains containing a conserved replication origin site (ARS1). Then fluorescently tagged MutSα and/or MutLα will be utilized, as previously described4, to determine whether they co-localize with the replication forks as they move along the DNA. Under normal circumstances, S. cerevisiae DNA polymerases make very few mistakes. To force the replication machinery into making more errors than normal a nucleotide imbalance will be tested. Every time the DNA polymerase makes a mistake that is not corrected by its 3? proofreading activity, the replication fork may continue working, or it may stop its progress and wait for the MMR system to correct the mistake. We will distinguish between these two possibilities using a single molecule technique. 1. Greene E, Wind S, Fazio T, Gorman J, Visnapuu M-L. DNA curtains for high-throughput single-molecule optical imaging. Methods Enzymol. 2010; 472: 293?315. 2. Silverstein T, Gibb B, Greene E. Visualizing protein movement on DNA at the single-molecule level using DNA curtains. DNA Repair. 2014; 20: 94?109. 3. Heller R, et al. Eukaryotic origin-dependent DNA replication in vitro reveals sequential action of DDK and S-CDK kinases. Cell. 2011; 146: 80?91. 4. Gorman J, et al. Single-molecule imaging reveals target-search mechanisms during DNA mismatch repair. Proc. Natl. Acad. Sci. U. S. A. 2012; 109: E3074?83.