C-terminal flap endonuclease (rad27) mutations: lethal interactions with a DNA ligase I mutation (cdc9-p) and suppression by proliferating cell nuclear antigen (POL30) in Saccharomyces cerevisiae.
During lagging-strand DNA replication in eukaryotic cells primers are removed from Okazaki fragments by the flap endonuclease and DNA ligase I joins nascent fragments. Both enzymes are brought to the replication fork by the sliding clamp proliferating cell nuclear antigen (PCNA). To understand the relationship among these three components, we have carried out a synthetic lethal screen with cdc9-p, a DNA ligase mutation with two substitutions (F43A/F44A) in its PCNA interaction domain. We recovered the flap endonuclease mutation rad27-K325* with a stop codon at residue 325. We created two additional rad27 alleles, rad27-A358* with a stop codon at residue 358 and rad27-pX8 with substitutions of all eight residues of the PCNA interaction domain. rad27-pX8 is temperature lethal and rad27-A358* grows slowly in combination with cdc9-p. Tests of mutation avoidance, DNA repair, and compatibility with DNA repair mutations showed that rad27-K325* confers severe phenotypes similar to rad27Delta, rad27-A358* confers mild phenotypes, and rad27-pX8 confers phenotypes intermediate between the other two alleles. High-copy expression of POL30 (PCNA) suppresses the canavanine mutation rate of all the rad27 alleles, including rad27Delta. These studies show the importance of the C terminus of the flap endonuclease in DNA replication and repair and, by virtue of the initial screen, show that this portion of the enzyme helps coordinate the entry of DNA ligase during Okazaki fragment maturation.
Karanja
Kenneth K
KK
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Livingston
Dennis M
DM
D034961:Antigens, Nuclear / Q000235:genetics; D002172:Canavanine / Q000494:pharmacology; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D025141:Drug Resistance, Fungal / Q000235:genetics; D004843:Epistasis, Genetic / Q000502:physiology; D045585:Flap Endonucleases / Q000737:chemistry / Q000235:genetics*; D005804:Genes, Lethal / Q000502:physiology; D005816:Genetic Complementation Test; D009154:Mutation / Q000502:physiology; D030781:Organisms, Genetically Modified; D010641:Phenotype; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013489:Suppression, Genetic / Q000502:physiology
D034961:Antigens, Nuclear; C000607890:CDC9 protein, S cerevisiae; C546594:POL30 protein, S cerevisiae; D018809:Proliferating Cell Nuclear Antigen; D029701:Saccharomyces cerevisiae Proteins; D002172:Canavanine; D045585:Flap Endonucleases; C468323:RAD27 protein, S cerevisiae; D011088:DNA Ligases; D000072481:DNA Ligase ATP