A study of protein phosphorylation and ubiquitination in DNA double strand break responses

Abstract

Through altering the properties of DNA damage response (DDR) proteins, posttranslational modifications (PTMs) play pivotal roles in orchestrating timely and accurate cellular responses to DNA damage. Amongst the PTM systems, protein phosphorylation and ubiquitination are two important regulatory mechanisms that drive mammalian DDRs, and they do so by controlling the activity of DDR proteins, their stability and their interaction with one another within the emerging DDR network. Accordingly, dysregulation of these crucial PTMs compromises DNA damage responses and contributes to genome instability. My PhD study was mainly focused on understanding how these two classes of PTMs promote DDRs.

In understanding the functional significance of breast cancer susceptibility gene product PALB2 in protecting genome integrity, I found that PALB2, as a key DNA repair factor and tumour suppressor, undergoes ATM-dependent phosphorylation at its Ser157 and Ser376 upon IR-induced DNA damage. Inhibiting PALB2 phosphorylation leads to sustained DDR activation. By systematically analyzing the regulation of PALB2 phosphorylation events, my study uncovers a new phosphorylation-dependent regulatory mechanism in protecting genome integrity.

Next, to systematically study protein ubiquitination in DNA damage responses, a small interference RNA (siRNA) library designed to target known and predicted E2 ubiquitin conjugating enzymes was utilized to screen new regulators of DNA double-strand break (DSB) responses. Using this approach, RAD6A and UBE2U were identified as two important players that promote chromatin responses at DSBs. The roles of these proteins in regulating DDRs were further characterized. Taken together, this study has uncovered novel ubiquitin components that participate in cell response to DNA damage.

Collectively, my study highlights important roles of PTMs in mammalian DDRs, and provides new leads to further appreciate the complexity and versatility of protein PTMs in genome stability maintenance and tumour suppression.