Lee Zou, Ph.D.
Center for Cancer Research, Mass General Research Institute
Professor of Pathology
Harvard Medical School
Jim and Ann Orr MGH Research Scholar (2011-2016)
Mass General Research Institute, Massachusetts General Hospital
|PhD Stony Brook Medicine 1999|
Genome. The genome is a dynamic and fragile structure constantly challenged by DNA damage, DNA replication problems, and other forms of cellular stress. Maintenance of genomic integrity is essential for the survival of all organisms.
In humans, loss of genomic integrity is closely linked to cancer, developmental defects, and aging.
The goal of our research is to elucidate the fundamental mechanisms by which cells inspect and protect the genome. The task of safeguarding genomic stability is not accomplished by a single cellular process. Instead, it relies on the integrated action of a number of processes including cell cycle progression, DNA replication, DNA repair, and others.
In the center of all these processes is a conductor called the checkpoint. The checkpoint is a complex signaling network evoked by DNA damage or genomic instability. Activated checkpoint regulates and coordinates many processes important for genomic stability.
The checkpoint is an important barrier against the genomic instability induced by oncogenic stresses. During early tumorigenesis, the checkpoint is activated and it hinders cancer progression by inducing senescence or apoptosis.
Mutations that impair the checkpoint (e.g. p53, Brca1, ATM, and Chk2) associate with both cancer predisposition and cancer progression. The ATR-ATRIP kinase is a master controller of the checkpoint. Elicited by a broad spectrum of DNA damage and replication interference, the ATR-mediated checkpoint is particularly important for genomic stability during genome duplication.
We are currently focusing on the mechanisms by which cells sense DNA damage and activate the ATR checkpoint, and the mechanisms by which the ATR checkpoint orchestrates DNA replication, DNA repair, and chromatin regulation.
Damage Sensing and Checkpoint Activation
How is checkpoint activated by DNA damage in cells? To address this question, we sought to identify the proteins sensing DNA damage as well as the DNA structures being sensed. Using both biochemical and cell biological approaches, we found that single-stranded DNA (ssDNA) coated with RPA, a common structure generated at DNA damage and stalled replication forks, is the key structure recognized by ATR-ATRIP.
The Rad17 and 9-1-1 (Rad9-Rad1-Hus1) complexes are two important regulators of ATR-ATRIP. Interestingly, these two checkpoint complexes are structurally related to RFC and PCNA, two DNA replication complexes recognizing specific DNA structures. We found that the Rad17 complex specifically recognizes the junctions of single- and double-stranded DNA in the presence of RPA, and it recruits 9-1-1 complexes onto DNA to activate ATR-mediated signaling.
Thus, our findings have revealed several key mechanisms used by the checkpoint to sense DNA damage. Importantly, our biochemical analyses have enabled us to establish an in vitro system 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 associating with DNA damage.
This system will be further explored to understand how DNA damage sensors initiate checkpoint signaling on DNA, and how DNA damage signals are relayed to downstream effectors.
Checkpoint and Replication Forks
The process of DNA replication is intimately linked to checkpoint signaling. During each S phase of the cell cycle, some elongating replication forks encounter various kinds of impediment or stress on chromatin, leading to activation of the replication checkpoint. The checkpoint activated at replication forks plays a crucial role in stabilizing the forks and allowing them to continue DNA synthesis.
Thus, the replication checkpoint is not only critical for the maintenance of genomic stability, but also essential for the complete duplication of the genome. Using both budding yeast and human cells as model systems, we are investigating how replication, checkpoint, and repair/recombination proteins function in concert at stressed replication forks.
Our goal is to understand how these intertwined cellular processes are orchestrated at elongating forks, and how replication elongation is regulated by the checkpoint on chromatin.
Checkpoint Signaling Beyond Protein Phosphorylation
The signaling of DNA damage through the checkpoint pathway is generally viewed as a cascade of protein phosphorylation events. However, several other types of protein modifications, such as ubiquitination, sumolation, methylation, and acetylation, are also regulated by DNA damage.
These protein modifications are implicated in many cellular processes regulated by the checkpoint pathway, but whether and how the checkpoint regulates these protein modifications remains largely unknown.
Furthermore, whether these protein modifications are important for signaling DNA damage through the checkpoint pathway is unclear. Using the ATR checkpoint as an example, we are now exploring how various protein modifications are implicated in checkpoint signaling and its downstream functions at replication forks.