Assessing the Role of Gap-Avoidance Mechanisms and Nucleosome Assembly Pathways as Guardians of DNA Replication Fork Stability
Open Access
- Author:
- Thakar, Tanay
- Graduate Program:
- Biomedical Sciences (PHD)
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 11, 2022
- Committee Members:
- Hong-Gang Wang, Outside Field Member
James Broach, Major Field Member
Zhonghua Gao, Major Field Member
Lisa Shantz, Outside Unit Member
George Moldovan, Chair & Dissertation Advisor
Ralph Keil, Program Head/Chair - Keywords:
- Replication Stress
Fork protection
Genome Stability
Nucleosome Assembly
PCNA
BRCA1
BRCA2 - Abstract:
- A global response to DNA replication stress is replication fork reversal, which involves the slowing and reversal of replication forks accompanied by the annealing of nascent DNA strands to form pseudo-four-way junctions. Functionally, reversal is thought to enable replication forks to avert DNA damage while replication stress is resolved. However, despite the protective functions of fork reversal, the annealed nascent arms of reversed forks a novel substrate that resembles one-end of a double stranded break, potentially susceptible to resection by milieu of nucleases such as CTIP, MRE11, EXO1 and DNA2. To prevent the undue processing of reversed replication forks, an extensive range of fork protection mechanisms are put into place. The most prominent of these mechanisms involves the Breast Cancer Susceptibility Proteins BRCA1 and BRCA2. Upon fork reversal, BRCA1 and BRCA2 directly influence the activity of the downstream effector protein RAD51, putatively by enabling it to form stable nucleofilaments on reversed fork substrates, which restricts the activity of nucleases and prevents the resection of stalled replication forks. In BRCA1/2 deficient cells CTIP, MRE11, EXO1 and DNA2 cooperate to elicit the long-range resection of forks that have undergone stalling and reversal. This long-range resection of reversed forks is associated with endonucleolytic DNA double strand break (DSB) induction and consequent genomic instability, thereby establishing fork protection as an essential component of BRCA-mediated tumor suppression. Critically, fork degradation also underlies sensitivity of BRCA-deficient cells to chemotherapeutic agents. As a corollary to this, the restoration of fork protection is associated with acquired chemoresistance in BRCA-deficient cancers. Therefore, gaining a better understanding of the fundamental determinants of fork protection is essential to gaining a better understanding of the etiology and therapeutic response of BRCA-deficient cancers. The first study reveals Proliferating Cell Nuclear Antigen (PCNA) K164-ubiquitination as a critical post-translational modification governing fork stability in a manner distinct from the BRCA-pathway. Through CRISPR/Cas9 mediated genome editing we create a homozygous PCNA-K164R mutation in human 293T and RPE-1 cells to render them completely devoid of PCNA ubiquitination. We show that PCNA-K164R cells are susceptible to DNA2-mediated fork degradation upon replication arrest by the ribonucleotide reductase inhibitor hydroxyurea (HU). We find that fork degradation in these cells is connected to their inability to mitigate replication associated gaps which interferes with Okazaki fragment (OF) synthesis and maturation, and consequently interferes with PCNA unloading from lagging strands. A direct result of this is the improper recycling of the PCNA-interacting histone chaperone CAF-1 which causes replication coupled nucleosome assembly defects, priming stalled forks for nucleolytic resection upon stalling and reversal. We further uncover a critical role for PCNA-ubiquitination in enabling the survival of BRCA-deficient cells upon treatment with PARP-inhibitors by suppressing single-stranded DNA (ssDNA) gaps, a novel therapeutic vulnerability in BRCA-deficient cancers. In the subsequent study we reveal an unexpected central role for nucleosome assembly in determining BRCA-dependent fork protection. We find inactivation of CHAF1A (the p150 subunit of CAF-1), through the activation of a compensatory replication-independent nucleosome assembly pathway dependent on the histone chaperone HIRA, restores fork stability to BRCA-deficient cells. Critically, we reveal ASF1-dependent nucleosome assembly as a general mechanism ensuring fork stability which operates in epistasis with the BRCA-pathway. Lastly, we elucidate the importance of BRCA-mediated replication associated gap suppression in ensuring CAF-1-dependent nucleosome assembly, thereby ensuring fork protection. We show that BRCA-deficient cells accumulate both leading and lagging strand ssDNA gaps during replication stress. Lagging strand gaps elicit PCNA unloading defects due to incomplete OF synthesis which result in a consequent CAF-1 recycling defect and impaired replication coupled nucleosome assembly. We therefore reveal CAF-1 as an essential effector of BRCA-mediated fork protection that operates through efficient replication associated gap suppression and PCNA unloading. These findings enable us to posit a model that unifies two major hallmarks of BRCA-deficiency in cancer, i.e., replication fork degradation and replication coupled ssDNA gap accumulation, into a sequential mechanism underlying genomic instability connected by PCNA-recycling and nucleosome assembly by CAF-1.