Biochemical analysis of PCNA ubiquitination reactions

Abstract

DNA is continuously insulted by not only endogenous metabolic products but also low levels of ionizing radiation and a wide variety of chemicals. Low levels of DNA damage does not disturb cell division cycles and cells complete DNA replication. Since DNA lesions usually inhibit DNA replication, cells have evolved mechanisms to restore stalled replication. One is translesion DNA synthesis (TLS), which can extend primer ends by specialized TLS polymerases using damage DNA as templates. The other is template switching (TS), in which primer ends are annealed with newly synthesized daughter strands as templates. TLS and TS are regulated by mono- and poly-ubiquitination of PCNA, respectively. Here, we report biochemical reactions of PCNA ubiquitination in vitro.