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Theses Doctoral

BRCA1 and 53BP1 Mediate Reprogramming Through DNA Repair Pathway Choice

Georgieva, Daniela Chavdarova

BRCA1 is a caretaker of genome integrity with various molecular functions, which are required for development and tumor suppression. These include the homology-directed repair (HDR) of DNA double strand breaks, stalled replication fork protection (SFP), transcription, chromatin remodeling and cell cycle checkpoint control. Recent studies reported that BRCA1 is required for reprogramming to pluripotency, but its specific role remains unknown. In this work, we use separation of function mutants for the roles of BRCA1 in HDR and SFP to show that BRCA1 is required to repair replication-associated DNA double strand breaks by homologous recombination during reprogramming. Deficiency in SFP proved inconsequential to induced pluripotent stem (iPS) cell generation and cells with this phenotype did not experience reduced reprogramming. Thus, the primary limiting factor for the transition to pluripotency is a specific class of DNA damage: double strand breaks, likely occurring in late replicating regions which require repair by homologous recombination.

These findings identify an important role of DNA damage, linked to the progression of DNA replication, in limiting cell type transitions during reprogramming. Most studies on iPS cell generation have focused on gene expression as a limiting step, in part due to the wide availability of tools to analyze transcription. Since the progression of DNA replication and DNA damage during S-phase are cell type specific, we have started the development of a sequencing platform to map various aspects of replication progression, such as origin usage, polymerase direction,pausing and stalling. In this work, we demonstrate that nucleotide analogs, incorporated during DNA synthesis in mammalian cells, can be detected by Nanopore sequencing.

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More About This Work

Academic Units
Cellular, Molecular and Biomedical Studies
Thesis Advisors
Egli, Dietrich Meinrad
Degree
Ph.D., Columbia University
Published Here
September 16, 2019