Brittany M. Ripley

Translesion synthesis is a major DNA damage bypass pathway in which specialized DNA polymerases, such as pol η and Rev1, are recruited to stalled replication forks where they catalyze nucleotide incorporation opposite DNA damage. Translesion synthesis is regulated by the Rad6-Rad18 complex, which catalyzes PCNA mono-ubiquitylation. Despite its central role in regulating translesion synthesis, its oligomeric state and molecular interactions are poorly understood. Here, we use mass photometry to show that Rad18 co-purifies with Rad6 and forms a series of larger oligomeric complexes. Using yeast two-hybrid studies, we showed that while the full-length Rad18 complex interacts with pol η, its interaction with Rev1 is controlled by auto-inhibition. The release of this auto-inhibition is associated with the dissociation of the Rad6-Rad18 complex, freeing Rad18 monomers and dimers. Because pol η participates in non-mutagenic translesion synthesis, while Rev1 participates in mutagenic translesion synthesis, this auto-inhibition likely allows pol η preferential access to stalled replication forks by delaying Rev1 recruitment. Such an ordered polymerase-recruitment mechanism would reduce the likelihood of mutations.

Summary: The ubiquitin-like protein, ISG15, can act as a cytokine or can covalently modify host and pathogen-derived proteins. The consequences of ISG15 modification on substrate fate remain unknown. Here we reveal that ISGylation of the Arp2/3 complex slows actin filament formation and stabilizes Arp2/3 dependent structures including cortical actin and lamella. When properly controlled, this serves as an antibacterial and antiviral host defense strategy to directly restrict actin-mediated pathogen spread. However, Listeria monocytogenes takes advantage in models of dysregulated ISGylation, leading to increased mortality due to augmented spread. The underlying molecular mechanism responsible for the ISG15-dependent impact on actin-based motility is due to failed bacterial separation after division. This promotes spread by enabling the formation of multi-headed bacterial “bazookas” with stabilized comet tails that can disseminate deeper into tissues. A bacterial mutant that cannot recruit Arp2/3 or a non-ISGylatable mutant of Arp3 is sufficient to rescue slowed comet tail speed and restrict spread. Importantly, ISG15-deficient neonatal mice have aberrant epidermal epithelia characterized by keratinocytes with diffuse cortical actin, which could underlie observed defects in wound healing in human patients who lack ISG15. Ultimately, our discovery links host innate immune responses to cytoskeletal dynamics with therapeutic implications for viral infection and metastasis.

DNA damage blocks the replication fork, which can result in fork collapse, single stranded breaks, and ultimately cell death. Translesion synthesis (TLS) is a pathway by which a special set of DNA polymerases, which include Rev1 and Pol η, are recruited to bypass the DNA lesion. While Pol η replicates past damage in an error-free manner, Rev1 and the helper polymerase, Pol ζ, are responsible for >90% of UV-induced mutations in cells. Regulation of these polymerases is essential to preventing genome instability due to the error-prone Rev1 pathway. One of the modes of TLS regulation is through polymerase recruitment to the damage. Rad6-18 recruits TLS polymerases via direct interactions with both Rev1 and Pol η. I found that Pol η binds Rad6-18 with its PCNA interacting peptide (PIP1) motif. In addition, I showed that both the PIP1 and the R6BD of Rad18 can bind Rad6 simultaneously. In addition, I found a second PIP-like motif (PIP2) which binds both PCNA and Rad6-18 with micromolar affinity and fast on and off rates. Furthermore, mutations to this PIP2 increases sensitivity to UV-induced DNA damage to a similar degree as PIP1 mutations. Additionally, I discovered that the interaction between Rad18 and Rev1 is autoinhibited by intramolecular interactions in Rad18. Furthermore, a separation of function point mutation on the semi-conserved residue, W387A, releases inhibition of Rev1 binding Rad18 without affecting Rad6 interaction with the R6BD. In conclusion, this data supports a model in which Rad18 tightly regulates TLS polymerase recruitment to the complex until absolutely needed to avoid high mutation rates.

DNA damage leads to genome instability by interfering with DNA replication. Cells possess several damage bypass pathways that mitigate the effects of DNA damage during replication. These pathways include translesion synthesis and template switching. These pathways are regulated largely through post-translational modifications of proliferating cell nuclear antigen (PCNA), an essential replication accessory factor. Mono-ubiquitylation of PCNA promotes translesion synthesis, and K63-linked poly-ubiquitylation promotes template switching. This article will discuss the mechanisms of how these post-translational modifications of PCNA control these bypass pathways from a structural and biochemical perspective. We will focus on the structure and function of the E3 ubiquitin ligases Rad18 and Rad5 that facilitate the mono-ubiquitylation and poly-ubiquitylation of PCNA, respectively. We conclude by reviewing alternative ideas about how these post-translational modifications of PCNA regulate the assembly of the multi-protein complexes that promote damage bypass pathways.