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.
Schmidt
Kristina H
KH
Ludwig Institute for Cancer Research, Cancer Center, and Department of Medicine, University of California San Diego School of Medicine, La Jolla, California 92093-0660, USA.
Derry
Katrina L
KL
Kolodner
Richard D
RD
D020816:Amino Acid Motifs; D001665:Binding Sites; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D017930:Genes, Reporter; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019098:Telomerase / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D018809:Proliferating Cell Nuclear Antigen; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C099024:EST1 protein, S cerevisiae; D019098:Telomerase; C459081:Rrm3 protein, S cerevisiae; D004265:DNA Helicases