1. Molecular mechanisms that suppress DNA replication defects and chromosomal instabilities

a. Replication fork stress caused by transcription-induced conflicts

Increasing evidence suggest that transcription can be a significant driver of genomic instability. Improper regulation of transcriptional process can impede DNA replication or repair. Conflicts between transcription and replication machineries (e.g. caused by RNA polymerase interfering replication process) is now considered to be an important source for instability of common fragile sites (CFSs), breakage-prone chromosomal loci that are intimately connected to genomic aberrations and cancer development. Cells have evolved mechanisms to suppress the Transcription-Replication Conflicts (TRC), but specific factors or spatio-temporal arrangements involved in mitigating such processes in eukaryotic cells are only beginning to be understood. We are investigating the roles of newly identified complex containing OTUD5 deubiquitinase and FACT (FACilitator of Transcription) histone chaperone, in mitigating TRC and preserving genomic integrity. FACT has emerged as an important regulator of major essential DNA metabolisms including replication, transcription, and repair, yet how its activity is naturally regulated is poorly understood. In addition, we propose that Polycomb group epigenetic silencers (e.g. BMI1, RNF2) play important roles in suppressing the transcription-induced replication fork instabilities. We aim to decipher how these trans-acting factors form a network to promote uninterrupted replication and suppress genomic aberrations and development of tumorigenic

b. Cellular response to DNA double strand breaks

DNA double strand break (DSB) is one of the most lethal and mutagenic forms of DNA damage. Cells can use repair pathways (homologous recombination and non-homologous end joining) to repair DSBs, or cells proceed to programmed cell death to prohibit transmission of faulty genetic materials. When not handled properly, DSBs are known to cause chromosome translocations, cancer, and other pathological conditions. Our lab investigates mechanisms and factors that promote proper processing of DSBs.  

c. Deciphering the roles of Fanconi Anemia-associated proteins in replication fork stability.

Fanconi Anemia (FA) is a genomic instability disorder that is associated with defective DNA repair activities. FA individuals display congenital anomaly, bone marrow disorder, and increased cancer incidence. At the cellular level, FA cells show hypersensitivity to DNA damaging agents such as Cisplatin and Mitomycin C. There are currently 19 FA subtypes (FANCA ~ FANCT) identified, whose gene products are believed to function in a common pathway that coordinate and execute DNA repair activities. Presence of such large number of protein subgroups that give rise to FA underscores the complex genetics of the disease and presents FA as an important model disease to study how DNA repair activities are executed in response to genotoxic insults. One of the underlying molecular events defective in the FA cells is monoubiquitination of FANCD2 protein by a group of nuclear FA proteins (FANC-A, B, C, E, F, G, L, and M) that function as an E3 ubiquitin ligase. The monoubiquitinated FANCD2 functionally interacts with other DNA repair proteins, such as RAD51 DNA homologous recombination (HR) repair factor, or nucleases FAN1 and CtIP, to facilitate DNA repair pathways in response to various genotoxic stresses. We are interested in understanding the regulation and function of FANCD2 monoubiquitination, as well as the functional interplay between the FA proteins and the canonical HR repair proteins. 

2. Identification of ubiquitin-proteasome machineries in tumor suppression and genomic stability.

The ubiquitin-proteasome system (UPS) is an essential regulatory mechanism for promoting genome stability. E3 ubiquitin ligases and deubiquitinating enzymes (DUBs), integral components of the UPS, have emerged as key players in the maintenance of the genome stability. Our lab particularly studies function and regulation of DUBs in promoting DNA replication fork maintenance and repair in human cells.

For these projects, we use a range of modern molecular biology tools that include mammalian cell culture, RNAi, gene knockouts, confocal microscopy, and mass spectrometry analysis.

Research keywords:

• Sanchez A, de Vivo A, Tonzi P, Kim J, Huang TT, Kee Y. Transcription-replication collision as a source of common fragile site instability caused by BMI1-RNF2 deficiency. PLOS Genetics. 2020 Mar 6;16(3)
• de Vivo A, Sanchez A, Yegres J, Kim J, Emly S, Kee Y. The OTUD5-UBR5 complex regulates FACT-mediated transcription at damaged chromatin. Nucleic Acids Research, 2019 Jan 25;47(2):729-74
• Sanchez A, de Vivo A, Uprety N, Kim J, Stevens SM Jr., Kee Y. The BMI1-UBR5 axis regulates transcription repression at damaged chromatin. ​Proc. Natl. Acad. Sci. USA. 2016 Oct 4;113(40):11243-11248
• Cukras S, Lee E, Palumbo E, Benavidez P, Moldovan GL, Kee Y. The USP1-UAF1 complex interacts with RAD51AP1 to promote homologous recombination repair. ​Cell Cycle 2016 Oct;15(19):2636-2646
• Kee Y*, Huang TT*. The role of deubiquitinating enzymes in DNA repair. Review. Mol Cell Biol. 2015 Dec 7;36(4):524-44. *co-correspondence
• Kee Y*, D’Andrea AD*. Molecular pathogenesis and clinical management of Fanconi Anemia. ​J. Clinic. Invest. 2012. 122(11):3799-806 *co-correspondence