AND ABSTRACT Ionizing radiation and chemical agents induce strand breaks in DNA, which give rise to mutations and chromosomal alterations that can cause cancer. One of the chief biological defenses against DNA strand breaks is Double-Strand Break (DSB) repair, a complicated family of biologic pathways. Some modes of DSB Repair can proceed with full restoration of the DNA sequence, but others result in significant sequence change or loss. Many important questions remain regarding biochemical requirements for pathway selection. Within these broader issues are more specific questions regarding the mechanism of recruitment of proteins that participate in some DSB repair pathways but not others. In baker's yeast (S. cerevisiae), Slx4 protein recruits any of several endonucleases to DSB sites, which either remove an extraneous nonhomologous stretch of DNA or incise a four-way junction of DNA (a Holliday Junction) as one of the last steps of the repair event. The endonucleases recruited by Slx4 include Rad1-Rad10, Mus81-Mms4 and Yen1, but the biochemical details governing the selection of one endonuclease over another are not understood in sufficient detail. It is becoming increasingly clear that the phase of the cell cycle plays a critical role in endonuclease access and engagement of the DSB site. This project will detail the role of Slx4 in recruitment of endonucleases to DSBs in the yeast S. cerevisiae, focusing on why the SLX4 gene is needed for recruitment of endonuclease Rad1-Rad10 in some phases of the cell cycle but not others.
The specific aims of this proposal are to: 1) determine whether cells that are not actively engaged in cell division (those in G1) require SLX4 for repair product formation, 2) investigate whether Rad1-Rad10 recruitment to several specific types of DSB sites depends on SLX4 only in S phase, 3) investigate whether checkpoint signal dampening by Slx4 plays a role in SLX4-dependent recruitment of Rad1-Rad10 in DSB repair in dividing cells and, 4) determine whether Rad1-Rad10 colocalizes with Mus81-Mms4 or Yen1 in last-minute DNA repair during the final moments of chromosome separation in cell division and if such localization is SLX4-dependent.
These aims will be investigated with a variety of experimental techniques, including a relatively novel fluorescence microscopy approach in which DSBs will be induced and their repair monitored by fluorescence imaging of convergent fluorescent signals. In vitro techniques such as quantitative PCR and Chromatin Immunoprecipitation will also be used to provide corroborating results. These experiments will address important questions regarding the genetic and biochemical requirements for recruitment of Rad1-Rad10, Mus81-Mms4 and Yen1 to DSB sites. All three nucleases are conserved in all eukaryotes including humans. Understanding the molecular basis for genome instability will inform our understanding of the causes of cancer and aging, and will be important for advancing clinical strategies to minimize human suffering.
The proposed research will increase our understanding of the mechanism of regulation of a family of complex biological pathways that protect against cancer and aging known as the Double-strand Break Repair pathways. Results of this work may aid in developing cancer prevention and therapy strategies.
