Maintenance of genomic integrity is essential for the survival of all organisms. In humans, loss of genomic integrity is closely linked to cancers and other genetic diseases. The genomes of cells are constantly challenged by DNA damage, DNA replication problems, and other forms of cellular stresses. In response to such stresses, cells activate a complex signaling pathway, termed the checkpoint, to orchestrate various cellular responses. The checkpoint-mediated regulation and coordination of processes such as cell cycle transitions, DNA replication, DNA repair, transcription, and apoptosis are crucial for the stability of the genome. Mutations impairing the functions of this signaling pathway associate with cancers and cancer predisposition syndromes (e.g. p53, Brca1, ATM, Chk2, and Nbs1). Furthermore, checkpoint is activated at the early stage of human tumorigensis, suggesting a role of checkpoint as an anti-cancer barrier against the genomic instability induced by oncogenic stresses.
The long-term goal of our research is to understand how checkpoint is activated by genomic instability and oncogenic stresses, and how it coordinates and integrates the network of cellular processes to preserve genomic stability. Currently, our research is focused on three fundamental questions about checkpoint signaling. First, how is checkpoint activated by DNA damage in cells? Second, how does checkpoint transmit DNA damage signals through different types of protein modifications? Third, how does checkpoint protect the DNA replication forks encountering DNA damage? To address these questions, we are developing new biochemical and cell biological assays to examine the functions of the key checkpoint proteins. The ATR-ATRIP kinase complex is a central player for the checkpoint responses in human cells. We recently found that single-stranded DNA (ssDNA) coated with RPA, a common structure generated at DNA damage and stalled replication forks, is the key structure that recruits ATR-ATRIP. Our biochemical analyses have enabled us to establish an in vitro assay recapitulating the initial steps of checkpoint activation. Using this system, we are systematically investigating how the checkpoint-signaling complex (so called the “checkosome”) is assembled on RPA-coated ssDNA and other DNA structures, and to identify novel proteins involved in checkpoint responses. We are also developing new assay systems to characterize stalled DNA replication forks in cells, and to visualize how replication and repair are coordinated. Furthermore, we are exploring new strategies to target cancer cells using specific checkpoint regulators as a weapon.
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