Checkpoint Kinase-Dependent Regulation of DNA Repair and Genome Instability in Breast Cancer
Abstract
DDB1, a component of a Cul4A ubiquitin ligase complex, promotes nucleotide excision repair (NER) and regulates DNA replication. We have investigated the role of human DDB1 in maintaining genome stability. DDB1-depleted cells accumulate DNA double strand breaks in widely dispersed regions throughout the genome and have activated ATM and ATR cell cycle checkpoints. Depletion of Cul4A yields similar phenotypes, indicating that an E3-ligase function of DDB1 is important for genome maintenance. In contrast, depletion of DDB2, XPA, or XPC does not cause activation of DNA damage checkpoints, indicating that defects in NER are not involved. One substrate of DDB1-Cul4A that is crucial for preventing genome instability is Cdt1. DDB1-depleted cells exhibit increased levels of Cdt1 protein and re-replication, despite containing other Cdt1 regulatory mechanisms. Accumulation of DNA damage, re-replication, and activation of checkpoint responses in DDB1-depleted cells requires entry into S-phase and is partially, but not completely, blocked by co-depletion of Cdt1. Therefore, DDB1 prevents DNA lesions from accumulating in replicating human cells, in part, by regulating Cdt1 degradation. Loss of DDB1 function also likely inactivates the other ubiquitin-dependent mechanism of Cdt1 destruction (SCFSkp2), since active checkpoints in DDB1-depleted cells inhibit CDK activity. Thus, our data indicate that DDB1 has at least two unique and essential functions in human cells to maintain genome integrity.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jun 01, 2007
- Accession Number
- ADA474916
Entities
People
- Courtney A. Lovejoy
Organizations
- Vanderbilt University Medical Center