The goal of the proposed project is to characterize the regulatory mechanisms of eukaryotic nuclear DNA replication and repair that have evolved to protect and maintain genome fidelity. This proposal aims to build on our exciting new results that show replication and repair-associated proteins are post-translationally modified by acetylation, regulating their enzymatic function. Our preliminary results suggest that acetylation of replication/repair-associated proteins alters their functional properties in a manner that promotes formation of longer flaps during lagging strand DNA replication and repair. We hypothesize that the acetylation-based promotion of long flaps ensures that DNA-based transactions within active chromatin proceeds with higher fidelity. This occurs in replication via removal of a longer section of the initiator primer that was synthesized by the error-prone DNA polymerase ?, or in repair via the replacement of a longer patch of damaged DNA. The three specific aims of the current grant proposal are specifically focused on elucidating the role of acetylation during various DNA transactions. The K99 mentored phase of the grant will be carried out in the laboratory and under the guidance of Dr. Robert A. Bambara.
In Aim 1, I will initially determine how acetylation alters the biochemical properties of each replication and repair protein and their interacting partners. Both purified human and yeast proteins will be assayed in vitro, so that acetylation-controlled functional changes in individual proteins can be correlated to functional regulation that occurs in a human or yeast cellular context, respectively, in Aim 3. Using mass spectrometric analysis, I will also determine the lysine sites that are being modified in vitro and compare them to the reported in vivo acetylome.
In Aim 2, Okazaki fragment processing and base excision repair will be reconstituted, and the effects of acetylation on long patch creation will be determined. Training during the mentored phase, under the guidance of my co-mentor, Dr. Jeffrey J. Hayes, to reconstitute DNA templates on nucleosomes will help me to test the effects of acetylation on replication and repair in a reconstituted chromatin environment. During the mentored phase, I will also take advantage of the expertise of Dr. Eric M. Phizicky, also a co-mentor on this grant application, to train in the field of yeast genetics. Learning to reconstitute replication and repair pathways on nucleosomal templates and manipulate genetic aspects of the yeast model system, will form the core of my technical training in the mentored phase, which will amply equip me to perform experiments outlined in Aim 3. Studies outlined in Aim 1 and the majority of the studies outlined in Aim 2 will be completed during the mentored phase of the grant. During my independent phase, I would like to direct a laboratory that works on elucidating the roles of post-translational modifications on replication/repair-associated proteins on DNA transactions within the chromatin environment. To this end, in Aim 3, I will build on my results from Aims 1 and 2 to understand how acetylation of replication and repair proteins work in the context of the cell, where other cellular factors and post-translational modifications work together to regulate replication and repair pathways. Modulation of DNA transactions by acetylation of non-histone proteins is a relatively new concept with far reaching outcomes. Using mammalian cell and yeast systems, we will determine whether replication/repair-associated proteins are acetylated in response to euchromatin or heterochromatin replication, different cell cycle phases, genetic alterations that reduce the fidelity of replication, or specific damaging agents. Our results will define the role of acetylation as a signal regulator that promotes use of a specific pathway to increase genome fidelity. Understanding the regulation of the replication and repair process is important because the errors that build up over decades in humans are a direct cause of the aging process, and they are also the basis of development of diseases such as cancer and neurodegenerative disorders. Enzymes regulating the protein modifications during DNA transactions can be used as novel targets to develop innovative therapies to prevent genome deterioration.
Proteins that are involved in DNA replication and repair are modulated by acetylation, which alters their functional characteristics and defines how they participate in specific pathways. Our work involves understanding how regulation of these proteins ensures the protection of the genomic blueprint and prevents aging-related diseases and cancer. Our results will clarify the role of acetylases that are involved in modulating the functions of replication/repair-associated proteins allowing for possible use of these enzymes as clinical targets to prevent loss of genomic information.
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