Every day, the integrity of the genome is threatened by intrinsic and exogenous sources of DNA damage. Such damage poses an especially great risk during DNA replication, as the high-fidelity replication machinery cannot bypass DNA lesions. To avoid double-stranded DNA breaks and genome destabilization, replication forks that encounter lesions trigger the recruitment of the DNA-damage response (DDR) machinery. Trans-lesion synthesis (TLS) is a major DDR pathway that facilitates the swap of a high-fidelity replicative polymerase with a Y-family TLS polymerase that can accommodate and bypass bulky lesions. Y-family polymerases are recruited to the replication fork following the monoubiquitination of the DNA sliding clamp, PCNA, by the Rad18/Rad6 complex (Rad18 is an E3 ubiquitin ligase, and Rad6 an E2 ubiquitin conjugating enzyme). Because TLS polymerases are error-prone, their recruitment must be tightly regulated to prevent unnecessary mutagenesis. With each new discovery it becomes increasingly clear that the machinery required for TLS initiation is dynamic, enigmatic, and multi-functional. I propose to elucidate the key interactions within the TLS initiation machinery.

Skills

• Protein purification 

• Size exclusion chromatography

• X-ray crystallography

• Molecular cloning

• Cryo-EM

• Isothermal titration calorimetry

• Microscale thermophoresis

• Mass photometry

• PyMol movie making

• Scientific communication