Analysis of the function of Mad2B in the mammalian cell cycle

Mad2B (also called Rev7 or Mad2L2) is a regulatory subunit of DNA polymerase ζ that is involved in translesion DNA synthesis (TLS). Previous studies have implicated Mad2B in several cell functions including cell cycle regulation where it was shown to inhibit the Anaphase Promoting Complex (APC)/cyclosome by binding to Cdc20/Cdh1. As Mad2B shares 26% identity and 48% similarity at the amino acid level with the human mitotic checkpoint protein Mad2 (also called Mad2A or Mad2L1), it has been suggested that Mad2B may also function as a mitotic checkpoint protein. However, recent studies have demonstrated that Mad2B has an important function in the DNA damage response in mammalian cells. Hence, it has been suggested that Mad2B may play a role in the DNA damage signaling pathway rather than in the spindle assembly checkpoint (SAC). The aim of this project was to investigate the function of Mad2B (Rev7) in mammalian cells. The results demonstrate that Mad2B is not a mitotic checkpoint protein. Instead the data indicate that Mad2B is involved in the DNA damage response. Interestingly, Mad2B binds to Cdc20 following DNA damage. Mad2B was also found to associate with Cdc27 (a component of the Anaphase Promoting Complex or APC/cyclosome) both before and after activation of the DNA damage checkpoint. In addition, the results of the in vitro ubiquitylation assay indicated that Mad2B does not inhibit APC/C activity. Taken together the results in this project suggest a model where binding of Cdc20 to the Mad2B (Rev3/Rev1)-APC/C complex following DNA damage may alter APC/C substrate specificity of as yet unidentified substrates.