title: Saccharomyces cerevisiae RRM3, a 5' to 3' DNA helicase, physically interacts with proliferating cell nuclear antigen.
abstract: 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.
authors.affiliation: 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.
authors.forename: Kristina H
authors.initials: KH
authors.lastname: Schmidt
authors.forename: Katrina L
authors.initials: KL
authors.lastname: Derry
authors.forename: Richard D
authors.initials: RD
authors.lastname: Kolodner
mesh_terms: 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
chemical_list: 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