title: Suppression of Saccharomyces cerevisiae rad27 null mutant phenotypes by the 5' nuclease domain of Escherichia coli DNA polymerase I.
abstract: RNA primer removal from Okazaki fragments during lagging-strand replication and the excision of damaged DNA bases requires the action of structure-specific nucleases, such as the mammalian flap endonuclease 1 (FEN-1). This nuclease contains two conserved motifs enriched with acidic amino acid residues that are important for catalytic function. Similar motifs have been identified in nucleases found in viruses, archebacteria, eubacteria, and in eukaryotes ranging from yeast to humans. Unique among these proteins, the putative FEN-1 homologue in Escherichia coli is contained within the N-terminal region of the DNA polymerase I (PolN). To demonstrate that the cellular functions of FEN-1 reside in PolN, we cloned and expressed the amino terminal domain (323 amino acid residues) of PolI in a Saccharomyces cerevisiae strain lacking the FEN-1 homologue RAD27. Overexpression of PolN suppressed, to varying degrees, phenotypes associated with a rad27 null strain. These include temperature sensitivity, Okazaki fragment processing, a mutator phenotype, a G2/M cell cycle arrest, minichromosome loss, and methyl methane sulfonate sensitivity. We purified Rad27 and PolN proteins in order to determine whether differences in their intrinsic nuclease activities or interaction with proliferating cell nuclear antigen (PCNA) could explain the partial suppression of some phenotypes. We found that the in vitro nuclease activities of Rad27 were more potent than those of PolN and the activity of Rad27, but not PolN, was stimulated by PCNA. We conclude that the N-terminal nuclease domain of E. coli polymerase I encodes a functional homologue of FEN-1.
authors.affiliation: Division of Molecular Medicine, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA 91010, USA.
authors.forename: Xuemin
authors.initials: X
authors.lastname: Sun
authors.forename: Phillis
authors.initials: P
authors.lastname: Wu
authors.forename: Li
authors.initials: L
authors.lastname: Zheng
authors.forename: Douglas
authors.initials: D
authors.lastname: Thrower
authors.forename: Arthur
authors.initials: A
authors.lastname: Partikian
authors.forename: Junzhuan
authors.initials: J
authors.lastname: Qiu
authors.forename: Binghui
authors.initials: B
authors.lastname: Shen
mesh_terms: D017124:Conserved Sequence; D004247:DNA / Q000494:pharmacology; D004256:DNA Polymerase I / Q000737:chemistry / Q000494:pharmacology*; D004261:DNA Replication; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D045585:Flap Endonucleases; D009154:Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins
chemical_list: C022262:Okazaki fragments; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D004256:DNA Polymerase I; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases; C468323:RAD27 protein, S cerevisiae