The process of DNA replication during S phase of the cell cycle is constantly challenged by the presence of damaged DNA on the replication template. Base lesions in chromosomes can cause DNA polymerase stalling, and if the stalled polymerase is not resolved than the replication fork will collapse, and the chromosome will be broken. Collapsed replication forks are, therefore, a serious threat to the maintenance of genome stability, and are thought to be a primary event in generating the genetic instability that allows normal cells to become cancer cells. In this project, we will focus on two important pathways that allow cells to tolerate DNA damage during S phase, the ATM and Rad3 related (ATR)- dependent replication checkpoint, and the DNA polymerase eta-dependent trans- lesion synthesis (TLS) damage bypass pathway. Recent results from my laboratory have revealed that these two pathways interact during a DNA damage response and, in particular, that pol eta can override the activation of ATR by DNA damage. In this project, we will focus on how ATR is activated by stalled forks, by studying the critical ATR activator TopBP1. We have found that TopBP1 senses the stalled fork, and that it recruits DNA polymerase alpha (pol 1) and the 911 complex to the stalled fork. Recruitment of these two factors by TopBP1 is required for ATR activation.
In Aim 1, we will investigate the molecular mechanism whereby TopBP1 senses stalled forks, and in Aim 2 we will probe the biochemical mechanism for how it then recruits pol 1 and 911.
In Aim 3, we will investigate how pol eta overrides the ATR response to DNA damage, and in Aim 4 we will investigate a novel, proteolytic-based mechanism that regulates pol eta function during the DNA damage response. If these goals are met, then we will have achieved a greater understanding of the molecular mechanisms involved in ATR activation, and in pol eta regulation. Importantly, we will have also increased out understanding of how the ATR and pol eta pathways interact, and this will allow for a more integrated view of how cells manage replication stress to emerge.

Public Health Relevance

DNA damage is a serious impediment to chromosome replication, a fundamental component of the process of cell division. When DNA damage is encountered during chromosome replication, it will stall the DNA polymerases that are responsible for duplication of the genetic material. This stalling can have severe consequences for the stability of the genome, as a stalled polymerase that is left unresolved can cause the replication process to collapse, and the chromosome to break. The repair of broken chromosomes can be imperfect, and can than thereby result in the chromosome translocations that are known to cause cancer. In this proposal, we focus on two cellular pathways that help cells deal with stalled polymerases. One is a signaling pathway, and we will study how this signaling pathway recognizes stalled polymerases and, how it is activated by them. The other pathway involves a specialized DNA polymerase that can replicate DNA even when it is damaged. We will study the regulation of this polymerase, and the ability of this polymerase to influence signaling that is derived from stalled replication. These studies will help us understand how cells manage stalled replication, and could form the basis for newer and more effective anti-cancer drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067735-10
Application #
8234213
Study Section
Special Emphasis Panel (ZRG1-GGG-A (90))
Program Officer
Reddy, Michael K
Project Start
2003-05-01
Project End
2015-01-31
Budget Start
2012-02-01
Budget End
2015-01-31
Support Year
10
Fiscal Year
2012
Total Cost
$317,552
Indirect Cost
$121,532
Name
University of Southern California
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Bai, Liping; Michael, W Matthew; Yan, Shan (2014) Importin ?-dependent nuclear import of TopBP1 in ATR-Chk1 checkpoint in Xenopus egg extracts. Cell Signal 26:857-67
Murphy, Christopher M; Michael, W Matthew (2013) Control of DNA replication by the nucleus/cytoplasm ratio in Xenopus. J Biol Chem 288:29382-93
Yan, Shan; Michael, W Matthew (2009) TopBP1 and DNA polymerase-alpha directly recruit the 9-1-1 complex to stalled DNA replication forks. J Cell Biol 184:793-804
Yan, Shan; Michael, W Matthew (2009) TopBP1 and DNA polymerase alpha-mediated recruitment of the 9-1-1 complex to stalled replication forks: implications for a replication restart-based mechanism for ATR checkpoint activation. Cell Cycle 8:2877-84
Kim, Seung-Hwan; Michael, W Matthew (2008) Regulated proteolysis of DNA polymerase eta during the DNA-damage response in C. elegans. Mol Cell 32:757-66
Kim, Seung-Hwan; Holway, Antonia H; Wolff, Suzanne et al. (2007) SMK-1/PPH-4.1-mediated silencing of the CHK-1 response to DNA damage in early C. elegans embryos. J Cell Biol 179:41-52
Holway, Antonia H; Kim, Seung-Hwan; La Volpe, Adriana et al. (2006) Checkpoint silencing during the DNA damage response in Caenorhabditis elegans embryos. J Cell Biol 172:999-1008
Yan, Shan; Lindsay, Howard D; Michael, W Matthew (2006) Direct requirement for Xmus101 in ATR-mediated phosphorylation of Claspin bound Chk1 during checkpoint signaling. J Cell Biol 173:181-6
Leach, Craig A; Michael, W Matthew (2005) Ubiquitin/SUMO modification of PCNA promotes replication fork progression in Xenopus laevis egg extracts. J Cell Biol 171:947-54
Holway, Antonia H; Hung, Crystal; Michael, W Matthew (2005) Systematic, RNA-interference-mediated identification of mus-101 modifier genes in Caenorhabditis elegans. Genetics 169:1451-60