During DNA replication, each cell copies three billion nucleotides/bases within a period of six-eight hours. A complex and well-coordinated network of proteins, work in tandem to ensure the successful completion of this process. The inactivation of any one these proteins, can lead to disastrous consequences like genomic instability, cancer and many other debilitating diseases. Accordingly, the deficiency in any one of nineteen such proteins results in a devastating disorder called Fanconi anemia (FA). FA is a rare genetic disorder characterized by bone marrow failure (BMF), hematological abnormalities and a very high incidence of malignancies. BMF is a leading cause of mortality in FA patients  and pancytopenia, thrombocytopenia and anemia are common in FA patients . In addition, the FA patients have a progressive decline in hematopoietic stem and progenitors cells (HSPC) [67, 69, 70], which is thought be responsible for the bone marrow failure in patients. However, the primary mechanisms underlying HSPC decline, BMF and cancer predisposition in FA patients have remained elusive. Over the last decade, an increasing number of proteins functioning in important cellular pathways are being classified as FA proteins, highlighting the complexity of FA. While the DNA repair-mediated functions of FA proteins are widely implicated in the etiology of the disease, there is a driving need to understand the entire spectrum of cellular functions of FA proteins outside crosslink repair. Recent reports suggest a new role for the FA pathway in DNA replication. The long-term goal of this proposal is to investigate defective replication as one of the early driving forces of genomic instability, leading to hematopoietic stem and progenitor cell attrition, in Fanconi anemia patients. The genome-wide role of the FA proteins in DNA replication will be better understood by investigating their involvement facilitating the replication of regions of the genome that are most vulnerable to replication stress, such as fragile sites. The primary goal of this proposal is to elucidate the mechanistic involvement of the FA proteins in DNA replication, using fragile sites as model genomic loci. Preliminary studies by Dr. Madireddy, show that the FA proteins, specifically FANCD2, facilitates the replication of common fragile sites, even in the absence of exogenous replicative stress. The focus of Aim 1 is to identify structural elements, which potentially stall replication forks, in the absence of FA proteins and to elucidate the mechanism/s by which FANCD2 alleviates replication pausing at CFS. To understand why DNA replication initiation is altered, in the absence of the FANCD2 protein, Aim 2 investigates the chromatin remodeling role of FANCD2 is this process. Finally, using FA iPSC and reprogrammed hematopoietic stem cells (HSC) as model systems, Aim 3 investigates whether replication defects and the associated genomic instability are contributing to stem cell attrition in the earliest stages of FA. We expect that these proposed studies will both greatly increase our understanding of the mechanistic involvement of the FA pathway in DNA replication and genomic instability, and allow us to establish new paradigms regarding the contribution of replicative defects to the etiology of Fanconi anemia.
Multiple cellular pathways play a pivotal role during DNA replication and DNA repair to ensure the faithful and timely duplication of the human genome, to prevent genomic instability and other diseases. The deficiency of proteins involved in the Fanconi anemia pathway lead to a spectrum of degenerative changes such as hematopoietic abnormalities, bone marrow failure and an increased predisposition to malignancies. This proposal examines the role of Fanconi anemia proteins in DNA replication and genomic instability and will advance our knowledge about molecular mechanisms that contribute to FA associated hematopoietic abnormalities and cancers, in an effort to provide new insights for future therapies.
|Madireddy, Advaitha; Gerhardt, Jeannine (2017) Replication Through Repetitive DNA Elements and Their Role in Human Diseases. Adv Exp Med Biol 1042:549-581|
|Pan, Xiaolei; Drosopoulos, William C; Sethi, Louisa et al. (2017) FANCM, BRCA1, and BLM cooperatively resolve the replication stress at the ALT telomeres. Proc Natl Acad Sci U S A 114:E5940-E5949|