In C. elegans , two apparent Cip/Kip family CKIs are encoded by the
cki-1 and
cki-2 genes found in tandem on chromosome II. Cki-1 and
cki-2 share 21-27% sequence identity with mammalian
p21 and
p27, and share 26% identity between themselves. Inactivation of
cki-2 by RNAi produces an impenetrant embryonic arrest phenotype. No apparent larval abnomalities were observed in the
cki-2 (RNAi) animals that hatch. It is unclear whether
cki-2 functions as a negative cell cycle regulator nor is it clear why
cki-2 (RNAi) is embryonic lethal. As one of several approaches to determine the role of
cki-2 , we tried a yeast two hybrid screen to identify interacting partners with the divergent
cki-2 C-terminus as bait. From 109 primary positives from 6X10 6 transformants, we got two true positives and sequenced them. Database searches revealed that cDNA sequences from the two positives were identical to the PCNA (Proliferating Cell Nuclear Antigen) coding sequence ( C. elegans cDNA W03D2.4). Following this, we mapped the PCNA binding site on CKI-2 so that we may be able to use this to block the CKI-2/PCNA interaction in vivo . The PCNA binding region of CKI-2 is located between Asn158 and Arg163 (NYMPVR). This sequence is very different from the conserved PCNA binding site used by other CKIs. This may mediate a novel regulatory mechanism of PCNA by CKI-2. We are now trying to identify the CKI-2 binding site on PCNA for comparative purposes. PCNA is an essential component of the DNA replication machinery, acting as the processivity factor for DNA polymerase d and e , while it is also required for nucleotide excision repair. PCNA interacts not only with DNA repair and replication enzymes, but also with cell cycle regulatory proteins such as
p21. This suggests that CKI-2 may mediate the coordination between cell cycle progression, DNA replication, and DNA repair. Loss of this important regulatory function may result in chromosomal / DNA damage at low frequency during early embryonic cell divisions when replication stress is high resulting in the
cki-2 (RNAi)-dependent embryonic arrest.