DNA damage is a major determinant of cell fate and thus poses a significant threat to organism health. On one hand, incorrect repair or replication of damaged DNA can result in accretion of mutations, referred to as genomic instability, which is considered an enabling characteristic of cancer. On the other hand, cancer therapy such as radiation and genotoxic chemotherapy, works by inducing lethal DNA damage in cancer cells. At the molecular level, DNA damaging agents cause DNA lesions or adducts that affect on or both strands of the DNA molecule. In proliferating cells, unrepaired DNA damage poses a major threat to genome stability as encountering DNA lesions arrests the progression of the high-fidelity replicative DNA polymerases. Prolonged stalling of the replication fork at these sites can lead to fork collapse and formation of double stranded DNA breaks (DSBs) which can cause cell death or initiate chromosomal translocations. To avoid this, fork stalling initiates a set of complex molecular events aimed at bypassing the lesion to quickly restore normal replication, without technically repairing the damage which is left behind to be fixed at a later time. At least three strategies have been described for restarting stalled forks (Fig. 1). Translesion synthesis (TLS) involves a switch from the replicative polymerase to one of the many low-fidelity TLS polymerases which can insert nucleotides across DNA lesions. This process is frequently mutagenic, as the original base on the template strand is unreadable by the TLS polymerases. To keep this mutagenic process under control, TLS is regulated so that it only occurs upon spatially and temporary restricted post-translational modification of the replication fork protein PCNA, which is a co-factor for replicative polymerases in its unmodified form, and for TLS polymerases in its mono-ubiquitinated form. Alternatively, in an error-free process, stalled forks can be restarted upon template switching (TS) which involves their regression by translocases such as ZRNAB3, annealing the two nascent strands, and allowing replication restart using the nascent strand of the unaffected sister chromatid as template. TS requires poly-ubiquitination of PCNA with K63-linked ubiquitin chains, which provide a docking platform for ZRANB3. Finally, a PCNA-independent process is also available, through homologous recombination (HR)-mediated engagement of the sister chromatid by the BRCA-RAD51 recombinase complex. The decision making process that regulates which one of this mutually-exclusive pathways is employed is not understood.
Our vision is to provide novel strategies for modulating the outcome of exposure to DNA damaging agents, in order to mitigate its detrimental impact on human health, by acquiring an intimate understanding of the molecular events occurring at stalled replication forks. To achieve this long-term goal, our research program encompasses three strategic themes:
- To uncover novel mechanisms that maintain fork stability during replication of damaged DNA, their regulation, and the key players involved in these processes (Molecular and cellular biology and Genomics).
- To infer how individual genetic make-ups affects the outcome of exposure to environmental mutagens (Personalized medicine and Functional genomics)
- To define novel targets in the DNA damage response to modulate the response to environmental DNA damaging agents (Chemical and molecular genetics)
Current and past research projects
1. PCNA and its post-translational modifications
PCNA is a homotrimeric ring-shaped protein that encircles DNA, conferring processivity to DNA polymerases during DNA replication. Post-translational modification of PCNA by ubiquitin and SUMO regulates fork restart and lesion bypass through mutagenic and non-mutagenic processes.
- Figure 2. PCNA and its modifications. A. Model of DNA replication showing PCNA at the replication fork (from Burges, 2009.) B. Front and side view of PCNA. Arrows indicate Lys 164. C. PCNA modifications: ubiquitination signals lesion bypass by translesion synthesis or template switching; SUMOylation inhibits HR by recruiting PARI (in mammals) or Srs2 (in yeast) (adapted from Bergink and Jentsch, 2009).
2. The role of mono-ADP-ribosylation in DNA repair.
Posttranslational modifications are widely used to regulate DNA repair processes. Poly-ADP-ribose polymerization is a type of post-translational modification catalyzed by PARP enzymes, with important roles in repair of single and double strand breaks. While the functions of PARP1 are well characterized, there are over 17 PARPs in human cells and most of them have not been characterized. Only recently it was shown that a subset of PARPs have mutations in the active site that abolishes poly-ADP-ribosyltranferase activity; instead, these enzymes can only catalyze the attachment of a single ADP-ribose molecule, a process termed mono-ADP-ribosylation. The role of this modification was not known, and in particular, it had not been associated with DNA repair previously. Our laboratory is the first to show that mono-ADP-ribosylation plays an essential role in DNA repair. We showed that the mono-ADP-ribosyltransferase PARP10 interacts with the PCNA, the master regulator of DNA replication, to promote replication of damaged DNA and DNA damage tolerance (Nicolae et al, J. Biol. Chem. 2014). We also found that the related ADP-ribosyltransferase PARP14 promotes double strand break repair by Homologous Recombination, through mono-ADP-ribosylation of RAD51 (Nicolae et al, Nucleic Acids Research 2015). Moreover, we recently showed that PARP10 mutation causes neurodegeneration in humans (Shahrour et al, Neurogenetics 2016). Finally, we found that PARP10 plays an essential role during carcinogenesis (Schlacher et al Nucleic Acids Research 2018). Our work described for the first time a previously unanticipated role for mono-ADP-ribosylation in DNA repair, and uncovered novel biological functions for the poorly characterized enzymes PARP10 and PARP14.
- Figure 3. PARP10 and PARP14 regulate PCNA-dependent fork restart. DNA damaging carcinogens (such as benzo[a]pyrene from tobacco smoke) form DNA lesions that block the progression of replicative DNA polymerases. Outcomes of fork arrest include both point mutations and chromosomal translocations, depending how fork restart mechanisms are engaged. Bypass of DNA adducts can occur through Translesion Synthesis (TLS), which employs specialized polymerases able to replicate through DNA lesions. Due to their intrinsically low fidelity, incorrect nucleotides can be inserted across the lesion. Upon arrest of the replication machinery at a DNA adduct, PCNA becomes ubiquitinated to specifically recruit TLS polymerases containing ubiquitin-recognition motifs. PARP10 regulates PCNA-dependent TLS. If the adduct is not bypassed, fork collapse can occur, resulting in DNA strand breaks. The Homologous Recombination (HR) machinery can use the sister chromatid to re-establish the replication fork. HR is generally error-free, and it is an essential mechanism protecting against carcinogenesis. Binding of the protein RAD51 to broken DNA is an essential step in the HR reaction, and we showed that PARP14 regulates this step.
3. Unraveling the mechanism of DNA damage-induced differentiation of myeloid leukemia cells.
DNA damage exposure is a major modifier of cell fate in both normal and cancer tissues. In response to DNA damage, myeloid leukemia cells activate a poorly understood terminal differentiation process. We found that the NFkB pathway directly activates expression of the proliferation inhibitor p21 in response to DNA damage in myeloid leukemia cells (Nicolae et al, Oncogene 2018). In order to understand the role of this unexpected regulatory event, we ablated the NFkB binding site we identified in the p21 promoter, using CRISPR/Cas9-mediated genome editing. We found that NFkB -mediated p21 activation controls DNA damage-induced myeloid differentiation. Moreover, we showed that PARI, a replisome protein involved in regulating DNA repair and replication stress, is overexpressed in myeloid leukemia cells, and its knockdown reduces leukemia cell proliferation in vitro and in vivoin xenograft mouse models (Nicolae et al, Oncogene 2019). PARI depletion enhances replication stress and DNA damage accumulation, coupled with increased myeloid differentiation. Mechanistically, we showed that PARI inhibits activation of the NFkB pathway. Finally, we showed that PARI expression negatively correlates with expression of differentiation markers in clinical myeloid leukemia samples, suggesting that targeting PARI may restore differentiation ability of leukemia cells and antagonize their proliferation. Our results uncover a p53-independent pathway for p21 activation involved in controlling hematopoietic cell fate.
- Figure 4. PARI suppresses replication stress in myeloid leukemia cells, which would otherwise result in DNA damage accumulation. DNA damage can activate the NFkB pathway to promote p53-independent expression of p21, resulting in cell cycle arrest and myeloid differentiation.
4. Novel mechanisms of PARP inhibitor resistance.
BRCA proteins are essential for Homologous Recombination DNA repair, and their germline or somatic inactivation is frequently observed in human tumors. Understanding the molecular mechanisms underlying the response of BRCA-deficient tumors to chemotherapy is paramount for developing improved personalized cancer therapies. While PARP inhibitors have been recently approved for treatment of BRCA-mutant breast and ovarian cancers, not all patients respond to this therapy, and resistance to these novel drugs remains a major clinical problem. Several mechanisms of chemoresistance in BRCA2-deficient cells have been identified. Rather than restoring normal recombination, these mechanisms result in stabilization of stalled replication forks, which can be subjected to degradation in BRCA2-mutated cells. We recently identified the first mechanism of resistance to PARP inhibitors which restores recombination in BRCA2-deficient cells (Clements et al, Nucleic Acids Research 2018). We showed that the transcriptional repressor E2F7 modulates the chemosensitivity of BRCA2-deficient cells. We found that BRCA2-deficient cells are less sensitive to PARP inhibitor and cisplatin treatment after E2F7 depletion. Moreover, we showed that the mechanism underlying this activity involves increased expression of RAD51, a target for E2F7-mediated transcriptional repression, which enhances both Homologous Recombination DNA repair, and replication fork stability in BRCA2-deficient cells. Our work describes a new mechanism of therapy resistance in BRCA2-deficient cells, and identifies E2F7 as a putative biomarker for tumor response to PARP inhibitor therapy.
- Figure 5. Model showing the impact of E2F7 on olaparib sensitivity of BRCA2-deficient cells. In wildtype cells, BRCA2-dependent loading of RAD51 to DNA breaks and stalled replication forks ensures correct HR and fork protection, which promote resistance to olaparib (A). In BRCA2-deficient cells, RAD51 loading is reduced and both HR and fork protection are impaired, which results in olaparib sensitivity (B). E2F7 represses expression of RAD51. Loss of E2F7 results in increased RAD51 levels, which allows more binding of RAD51 to damaged DNA. This promotes both HR and fork protection, restoring resistance to olaparib (C).
5. Impact of germline mutations in genome stability factors on human health.
While inherited deficiencies in DNA repair genes have been indisputably linked to cancer predisposition, the impact of DNA and chromatin stability factors on organism development in humans is less clear. In collaboration with Dr. Orly Elpeleg (Hadassah Medical Center, Israel) we showed that germline mutations in such factors, including PARP10 (Shahrour et al, Neurogenetics 2016), UBTF (Edvardson et al, American Journal of Human Genetics 2018), and RNF13 (Edvardson et al, American Journal of Human Genetics 2019) in human individuals are associated with severe neurodevelopmental disorders. These findings underscore the essential role of genome and chromatin stability during development.
6. Novel genomic methods to investigate structural genomic instability
While deep next generation sequencing is ideal for identifying point mutations, structural, translocations and other structural variations are difficult to detect using this technology since the reduced size of the reads (50-200bp) makes it statistically unlikely to chance upon translocation breakpoints. In contrasty, optical genome mapping using the Bionano Saphyr platform allows mapping of megabase-sized DNA, based on imaging of DNA fibers labeled with sequence-specific fluorescent tags. We recently employed this technology to investigate structural genomic variation in myeloid leukemia patients. (Xu et al, BioRvix 2019). We are currently using this approach to investigate mechanisms of genomic instability induced by DNA damage exposure.
- Figure 6. Workflow for SV detection by optical mapping using the BionanoSaphyrplatform. Samples from a myeloid leukemia patient show previously undetected structural variations, which are missing in matched normal cells.
7. HUWE1 and replication stress.
We recently found that the HUWE1 ubiquitin ligase is recruited to stalled replication forks by the replication factor PCNA. We found that HUWE1 contains a PCNA interacting domain (PIP-box) required for PCNA interaction. HUWE1-knockout cells show increased replication stress, which can be corrected by re-
expression of wild-type, but not PCNA interaction-deficient HUWE1 mutant. Importantly, we found that HUWE1 mono-ubiquitinates H2AX to promote gH2AX signaling, recruitment of repair proteins, and restart of stalled replication forks. Our work unexpectedly uncovered HUWE1 as a novel E3 ubiquitin ligase for H2AX, responsible for the efficient response to replication stress (Choe et al, EMBO Reports 2016).
- Figure 7. HUWE1 is recruited by PCNA to stalled replication forks to ubiquitinate H2AX (adapted from Coleman and Huang, News and Views, EMBO Reports 2016.)
Research Grants Support:
2019-2023 NIH –NIGMS 1R01GM134681 (MPI: Bielinsky, Moldovan)
2016-2021 NIH – NIEHS 1R01ES026184 (PI: Moldovan)
2019-2020 Pennsylvania Dept. of Health TSF CURE award
2015-2020 St. Baldrick Scholar Award from the St. Baldrick Foundation for Cancer Research
2018-2019 4Diamonds Pediatric Cancer Collaborative Research Award
2015-2018 Department of Defense (DoD) Peer Reviewed Cancer Research Program (PRCRP) Career Development Award
2014-2016 Conquer Cancer Award from the Concern Foundation for Cancer Research
2013-2015 V Scholar Award from the Jimmy V Foundation for Cancer Research
2014-2015 Jake Gittlen Foundation Collaborative Cancer Research Award