Localization of Y-Family Polymerases and the DNA Polymerase Switch in Mammalian Cells

During translesion synthesis past sites of damaged DNA, specialized Y-family polymerases are employed by the cell to replace the high stringency replicative polymerases and synthesize DNA past the damaged site. These polymerases are localized in replication factories during the S phase of the cell cycle. When progress of the replication fork is blocked, the polymerase accessory protein, proliferating cell nuclear antigen (PCNA), becomes ubiquitinated and the monoubiquitinated PCNA has an increased affinity for Y-family DNA polymerase η (polη). This chapter describes methods for visualizing the polymerases in replication factories, for analyzing the ubiquitination status of PCNA, and for measuring its interaction with polη in chromatin extracts.