Studies on DNA Replication, Repair, and Mutagenesis in Eukaryotic and Prokaryotic Cells

A comprehensive review of Escherichia coli DNA replication

In collaboration with Krystian Lazowski and Iwona Fijalkowska, we reviewed recent research on Escherichia coli DNA replication that paved the groundwork for many breakthrough discoveries with important implications for our understanding of human molecular biology, given the high level of conservation of key molecular processes involved. To this day, the research attracts much attention, partially because it is an important model organism, but also because the understanding of factors influencing replication fidelity might be important for studies on the emergence of antibiotic resistance. Importantly, the wide access to high-resolution single-molecule and live-cell imaging, whole-genome sequencing, and cryo-electron microscopy techniques, which were greatly popularized in the last decade, allowed us to revisit certain assumptions about the replisomes, and offered us very detailed insight into how they work. For many parts of the replisome, step-by-step mechanisms have been reconstituted, and some new players identified. Our comprehensive review summarized the latest developments in the area, focusing on (a) the structure of the replisome and mechanisms of action of its components, (b) organization of replisome transactions and repair, (c) replisome dynamics, and (d) factors influencing the base and sugar fidelity of DNA synthesis.

Stabilization of DNA polymerase iota in human cells

In collaboration with Justyna McIntyre, we studied the stability of DNA polymerase iota (Poliota) in human cells. Poliota belongs to the Y-family of specialized DNA polymerases engaged in the DNA–damage tolerance pathway of translesion DNA synthesis that is crucial to the maintenance of genome integrity. The extreme infidelity of Poliota and the fact that both its up- and down-regulation correlate with various cancers indicate that Poliota expression and access to the replication fork should be strictly controlled. In our studies, we identified RNF2, an E3 ubiquitin ligase, as a new interacting partner of Poliota, which is responsible for Poliota stabilization in vivo. Interestingly, while we reported that RNF2 does not directly ubiquitinate Poliota, inhibition of the E3 ubiquitin ligase activity of RNF2 affects the cellular level of Poliota, thereby protecting it from destabilization. Additionally, we indicated that this mechanism is more general, as DNA polymerase eta, another Y-family polymerase and the closest paralog of Poliota, share similar features.

Interaction between Y family polymerases and Rad23A and Rad23B in human cells

In collaboration with Irina Bezsonova, we demonstrated a novel interaction between each Y-family polymerase and the nucleotide excision repair (NER) proteins RAD23A and RAD23B. We initially focused on the interaction between RAD23A and Poliota, and through a series of biochemical, cell-based, and structural assays, we found that the RAD23A ubiquitin-binding domains (UBA1 and UBA2) interact with separate sites within the Poliota catalytic domain. While this interaction involves the ubiquitin-binding cleft of UBA2, Poliota interacts with a distinct surface on UBA1. We further found that mutating or deleting either UBA domain disrupts the RAD23A–Poliota interaction, demonstrating that both interactions are necessary for stable binding. We also provided evidence that both RAD23 proteins interact with Poliota in a similar manner, as well as with each of the Y-family polymerases. These results shed light on the interplay between the different functions of the RAD23 proteins and revealed novel binding partners for the Y-family TLS polymerases.

Role(s) of DNA polymerase iota and kappa in the resistance to temozolomide in glioblastoma brain tumors

In collaboration with Carlos Menck, we investigated role(s) of DNA polymerase iota and kappa in the resistance to temozolomide (TMZ) in glioblastoma (GBM) brain tumors that are associated with poor patient survival. The current standard treatment involves invasive surgery, radiotherapy, and chemotherapy employing TMZ. Resistance to TMZ is, however, a major challenge. Previous work from Carlos Menck’s group identified candidate genes linked to TMZ resistance, including genes encoding translesion-synthesis (TLS) DNA polymerases iota (Poliota) and kappa (Polkappa). We further investigated the roles of Poliota and Polkappa in TMZ resistance by employing MGMT (O-6-methylguanine-DNA methyltransferase)–deficient U251–MG glioblastoma cells, with knockouts of either POLI or POLK genes encoding Poliota and Polkappa, respectively, and assessed their viability and genotoxic stress responses upon subsequent TMZ treatment. Cells lacking either of these polymerases exhibited a significant reduction in viability following TMZ treatment compared with parental counterparts. The restoration of the missing polymerase led to a recovery of cell viability. Furthermore, knockout cells displayed elevated cell-cycle arrest, mainly in late S-phase, and lower levels of genotoxic stress after TMZ treatment, as assessed by a reduction in foci of the phosphorylated histone gamma-H2AX and flow cytometry data. This implied that TMZ treatment does not trigger a significant H2AX phosphorylation response in the absence of these proteins. Interestingly, combining TMZ with Mirin (a double-strand break repair-pathway inhibitor) further reduced the cell viability and increased DNA damage and gamma-H2AX–positive cells in TLS KO cells, but not in parental cells. Our findings underscored the crucial roles of Poliota and Polkappa in conferring TMZ resistance and the potential backup role of homologous recombination in the absence of these TLS polymerases. We hypothesized that targeting these TLS enzymes, along with double-strand break DNA–repair inhibition, could therefore provide a promising strategy to enhance TMZ’s effectiveness in treating GBM.