Saccharomyces cerevisiae RRM3, a 5′ to 3′ DNA helicase, physically interacts with proliferating cell nuclear antigen

Proliferating cell nuclear antigen (PCNA) plays an essential role in eukaryotic DNA replication, and numerous DNA replication proteins have been found to interact with PCNA through a conserved eight-amino acid motif called the PIP-box. We have searched the genome of the yeast Saccharomyces cerevisiae for open reading frames that encode proteins with putative PIP-boxes and initiated testing of 135 novel candidates for their ability to interact with PCNA-conjugated agarose beads. The first new PCNA-binding protein identified in this manner is the 5' to 3' DNA helicase RRM3. Yeast two-hybrid tests show that N-terminal deletions of RRM3, which remove the PIP-box but leave the helicase motifs intact, abolish the interaction with PCNA. In addition, mutating the two phenylalanine residues in the PIP-box to alanine or aspartic acid reduces binding to PCNA, confirming that the PIP-box in RRM3 is responsible for interaction with PCNA. The results presented here suggest that the RRM3 helicase functions at the replication fork.