DNA synthesis is essential during every cell cycle, but it is an intrinsically hazardous event because elongating replication forks can stall or collapse at sites of DNA damage or at protein obstacles. Collapsed forks impede complete genome duplication and activate DNA damage surveillance mechanisms that halt cell cycle progression; an alternative to these scenarios is the activation of recombination events. Thus, agents that disrupt DNA replication forks generally result in dangerous chromosomal translocations or other abnormalities. Although multiple proteins have been implicated in the maintenance of replication fork stability, many aspects of how stalled replication forks are maintained and restarted remain poorly understood.
A highly conserved eukaryotic histone deposition protein termed Asf1 contributes to heterochromatin formation and DNA damage resistance. Funding from the past grant period supported our solution of the three-dimensional structure of the globular domain of budding yeast Asf1. These data were used to guide extensive structure-function analyses, which defined two separate interaction surfaces on Asf1; one for recognition of the HIR chromatin assembly complex, and a different surface for histone recognition. Additionally, a new biological role for Asf1 was discovered: Asf1 is required for the maintenance of DNA replication proteins at stalled forks. Asf1''s role in genome stability depends on its ability to bind histones and promote acetylation of histone H3 on lysine 56 during S phase. In sum, previous work of this lab demonstrated that Asf1 is at the center of a highly regulated pathway that promotes replication fork stability by ensuring deposition of specifically modified histones.
In this project, experiments will address how Asf1 and H3-K56 acetylation affect the DNA structure, protein composition, and kinetic properties of DNA replication forks. These experiments will utilize a variety of biochemical and genetic techniques, focused on detecting aberrant nucleic acid structures and patterns of protein accumulation. For example, replication intermediates will be examined via two-dimensional and alkaline gel electrophoresis to detect broken forks, aberrant recombination intermediates, or nascent strand alterations. Molecular combing of halogen-labeled DNA will be used to assess the efficiency of replication fork movement and restart in the presence of damage. Chromatin immunoprecipitation used to determine whether abnormal recruitment of helicases, nucleases or recombination proteins occurs, and modified in vivo footprinting techniques will be used to test for evidence of loss of leading and lagging strand coordination at the mutant forks.
Broader Impact These experiments will have a wide scientific impact that is not restricted to the study of the yeast Asf1 protein. Asf1 is highly conserved throughout eukaryotic organisms, and serves as the intersection between the histone deposition machinery and the signaling pathway that monitors DNA damage. Therefore, the analyses of Asf1 proposed here will be instructive for understanding DNA replication, DNA repair and nucleosome formation in all eukaryotes. Additionally, we are developing several methodologies for analysis of nascent DNA strands and in vivo protein footprinting at replication forks in order to extend the range of tools available for study of these important structures.
This project also includes a significant educational component, with most of the funding used for research training of laboratory personnel, including funding for lab members to present their data at scientific meetings and via published literature. Furthermore, in previous years, five different undergraduates in this laboratory have co-authored publications resulting from their projects, and we are continuing with our tradition of prioritizing undergraduate research experiences as an integral part of our research via the NSF Research Experience for Undergraduates (REU) program.
In addition to training of undergraduate researchers, in the next year my laboratory will participate in educational opportunities for local high school students. For the last 7 years, a group of four UMMS faculty have been involved in teaching laboratory exercises for an Advanced Placement Biology Course at North High School (Worcester, MA). This has been a rewarding experience for the high school students and provides an opportunity for faculty to interact with and influence nascent scientists and increase participation in community outreach at UMMS.
