Regulation of PCNA mono-ubiqutination by FANCD2 and Rad51

Assoc Rsrch Scientist Pediatrics
15 York St, New Haven, CT 06510-3221

FANCD2 is a key player in FA pathway.  Besides core complex of FA pathway the function of FANCD2 was also regulated by proteins of other DNA repair pathway.  It has been shown that PCNA and Rad18 can interact with FANCD2 and regulate FANCD2 momo-ubiquitination. 

The Rad18 is an ubiquitin ligase responsible for PCNA mono-ubiquitination, which is very important for PCNA function in translesion synthesis.  We found that FANCD2, Rad51, Rad18 and PCNA may form a complex.  And in response to DNA damage agent hyxdroxyurea (HU) the interactions of these proteins are enhanced.  We also found that FANCD2 is required for increased interaction between Rad51 and Rad18, indicating that FANCD2 may be an important component in Rad51 and Rad18 complex formation.  The interaction of Rad51 and Rad18 is also partially increased in FANCD2 mono-ubiquitination deficient mutant cells, indicating that non-ubiquitinated FANCD2 is also functional in the complex formation. In addition, the PCNA mono-ubiqutination is greatly increased in response to HU.  This ubiquitination was impaired in FANCD2 deficient cells and FANCD2 siRNA knock down cells.  FANCD2 mono-ubiquitination deficient mutant partially rescued PCNA mono-ubiquitination.  The partial mono-ubiquitination of PCNA in response to HU in FANCA deficient mutant confirmed the role of non-ubiquitinated FANCD2 in PCNA mono-ubiquitination.  The normal mono-ubiquitination of PCNA in FANCJ deficient mutant demonstrated that the effect of FANCD2 in PCNA mon-ubiquitination is not due to FA pathway deficiency.  Rad51 was also involved in regulating PCNA mono-ubiquitination in response to HU.  Rad51 siRNA knock down cells are impaired on PCNA mono-ubiquitination in response to HU.  The role of Rad51 in regulating PCNA mono-ubiquitination did not require BRCA1, indicating that this function is independent of homologous recombination (HR).    More importantly FANCD2 deficient cells were hypersensitive to HU, whereas FANCD2 mono-ubiquitination deficient mutant cells, FANCD2 corrected cells, FANCA deficient cells and FANCJ deficient cells were not hypersensitive to HU.  Our data indicate that FANCD2 plays an important role in PCNA mono-ubiquitination and translesion synthesis partially in a mono-ubiquitination independent manner.  Rad51 also plays an important role in PCNA mono-ubiquitination and translesion synthesis in a HR independent fashion.