Does the BLM Gene Co-regulate BRCA1 in DNA Damage Response?

Institution: Lawrence Berkeley National Laboratory
Investigator(s): Albert Davalos, Ph.D. -
Award Cycle: 2002 (Cycle VIII) Grant #: 8FB-0148 Award: $80,671
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2002)
Several years ago, a model was proposed that divided cancer susceptibility genes into two categories, those that (i) prevented inappropriate cell growth, and (ii) protected the genome. The first were termed "gatekeepers", which reflected their ability to prevent uncontrolled growth. These genes often are classical tumor suppressor genes like the Retinoblastoma gene Rb. The second class of genes was called "caretakers", which serve to maintain normal function of the "gatekeeper" genes. BRCA1 and BLM (i.e, the gene mutated in Bloom's syndrome (BS), which has DNA helicase activity) are considered "gatekeeper" genes. Yet, how they preserve the genome is not well understood. Cells from persons with Bloom's syndrome display excessive numbers of DNA exchanges, both between and within chromosomes, as well as a high frequency of somatic mutations. This project examines the critical role that BLM may play in the regulation of BRCA1 and directly examines whether the inactivation of BLM may contribute to breast cancer.

Our preliminary results, obtained in fibroblasts, indicate that BLM prevents cell death when cells encounter DNA damage during replication of the genome. The first project aim will examine whether inactivation of BLM in mammary epithelial cells results in drug hypersensitivity similar to observed in BS fibroblasts. A central question in this aim is how BLM and BRCA1 interact to maintain genomic stability. To test this question we will abolish BLM activity in mammary epithelial cells through retroviral transfer of DN BLM. We will compare the response of wt and BLM-deficient epithelial cells to DNA damage during S-phase. Also, investigate whether loss of both BLM and BRCA1 leads to a synergistic response to DNA damage. The second project aim will examine proteins that may modify BLM and BRCA1, and which may be required for their interaction and/or function. Using both in vitro and in vivo biochemical assays, we will examine whether BLM is a substrate for ATM or/and ATR, and what role phosphorylation plays in BLM and BRCA1 interactions. The last aim tests our hypothesis in an animal model of breast tumor progression. Utilizing nude mouse, examine whether loss of functional BLM drives oncogenically "primed" mammary epithelial cells to form tumors following injection; and whether co-injection of senescent fibroblasts accelerates tumor size and decreases latency.

Breast cancer progression occurs through dysregulation of numerous gene products. Therefore, central to understanding the etiology of breast cancer is to decipher how cells maintain an intact genome. Unique reagents available in our laboratory allows us to closely examine the role BLM may play in regulating BRCA1 as well as other proteins implicated in repairing damage during DNA replication.


Final Report (2004)
This project investigated whether the RecQ helicase, called BLM, regulates the breast cancer susceptibility gene BRCA1. Also, we explored the role BLM plays in maintaining genomic stability, since genomic integrity is a key component in preventing breast tumorigenesis. Our results demonstrate that the BLM protein responds rapidly to chromosomal double strand breaks caused by collapse of replication forks. Moreover, we observed in Bloom syndrome (BS) fibroblasts that the absence of wild type BLM protein prevents early recruitment of BRCA1 and NBS1 (a component of the key MRE11/Rad51/NBS1 repair complex) proteins to sites of double strand breaks induced by DNA replication perturbations. Importantly, BLM moves to sites of sites of double strand breaks in mammary epithelial cells that express mutant BRCA1 or cells deficient in NBS protein. Thus, BLM acts “upstream” of BRCA1 and NBS1 when cells encounter stress during replication of their genome.

During this project, other investigators showed that NBS 1 and BRCA1 may assist in the activation of regulatory proteins. Our data suggest exposure of cells to prolonged replication stress produces activation of the signaling kinase ATM and aggregation of the adaptor protein 53BP1 (note: 53BP1 localizes to double-strand breaks following irradiation, indicating that it might be a checkpoint protein) at sites of presumed collapsed replication forks. However, cells that lack functional BLM exhibit aberrant ATM activation and fail to recruit 53BP1 to sites of replication stress-induced damage. When cells are exposed to drugs that damage DNA irrespective of replication, proper activation and aggregation of both proteins occurs. Significantly, inactivation of the tumor suppressor protein p53 restored timely localization of both proteins to sites of replication stress. Taken together, our work suggests that p53 acts in concert with the BLM protein to modulate cellular activities after replication stress.

The signaling proteins ATM and ATR modify many repair proteins, which affects their movement and activity during the repair of a damaged genome. Our research suggests that ATR and ATM are necessary for BLM to move from an endogenous sub-nuclear location, marked by the PML protein to sites of replication stress-induced damage. Moreover, we observe that the 3 signaling proteins ATM, ATR and DNA PK (DNA-dependent protein kinase) become activated following drug-induced replication stress. This data suggest that either multiple repair mechanisms repair genomic damage due to replication stress or each protein plays a role in the faithful repair of the damage.

In conclusion, we have established reagents that will now allow us to more closely look at how DNA repair “caretaker proteins”, like BRCA1, BLM, NBS 1, operate in concert with p53 to protect mammary epithelial cells from undergoing transformation when their genome encounter problems during replication. Our goal is to explore the damage response of human mammary epithelial cells in a 3-D cell culture system that more closely mimics normal tissue structures. We expect that this system combined with the information developed in the project will have the potential to uncover biological markers that could aid in earlier detection of very early transformed breast cells.

Bloom syndrome cells undergo p53-dependent apoptosis and delayed assembly of BRCA1 and NBS1 repair complexes at stalled replication forks.
Periodical:Journal of Biological Chemistry
Index Medicus: J Biol Chem
Authors: Davalos AR, Campisi J
Yr: 2003 Vol: 162 Nbr: 7 Abs: Pg:1197-209

ATR and ATM-dependent movement of BLM helicase during replication stress ensures optimal ATM activation and 53BP1 focus formation.
Periodical:Cell Cycle
Index Medicus: Cell Cycle
Authors: Davalos AR, Kaminker P, Hansen RK, Campisi J
Yr: 2004 Vol: 3 Nbr: 12 Abs: Pg:1579-86

Telomere length mediates the effects of telomerase on the cellular response to genotoxic stress.
Periodical:Experimental Cell Research
Index Medicus: Exp Cell Res
Authors: Rubio MA, Davalos AR, Campisi J.
Yr: 2004 Vol: 298 Nbr: 1 Abs: Pg:17-27