Mgs1 and Rad18/Rad5/Mms2 are required for survival of Saccharomyces cerevisiae mutants with novel temperature/cold sensitive alleles of the DNA polymerase delta subunit, Pol31.
Saccharomyces cerevisiae DNA polymerase delta (Pol delta) is a heterotrimeric enzyme consisting of Pol3 (the catalytic subunit), Pol31 and Pol32. New pol31 alleles were constructed by introducing mutations into conserved amino acid residues in all 10 identified regions of Pol31. Six novel temperature-sensitive (ts) or cold-sensitive (cs) alleles, carrying mutations in regions III, IV, VII, VIII or IX, conferred a range of defects in the response to replication stress or DNA damage. Deletion of SGS1, RAD52, SRS2, MRC1 or RAD24 had a deleterious effect only in combination with those pol31 alleles that had a phenotype as single mutants, suggesting a requirement for recombination and checkpoint functions in processing the DNA lesions or structures that form as a consequence of replication with a defective Pol delta. In contrast, deletion of POL32 negatively affected the growth of almost all pol31 mutants, suggesting an important role for all conserved amino acids of Pol31 in maintaining the integrity of Pol delta complex structurally, at least in the absence of the third subunit. Surprisingly, deletions of RAD18 and MGS1 aggravated the temperature sensitivity conferred by most ts or cs alleles and specifically suppressed the hys2-1 and hys2-1-like mutations of POL31. Deletion of RAD5 or MMS2 had an effect on pol31 ts/cs mutants similar to that of RAD18, whereas deletion of RAD30 or REV3 had no effect. We propose that Rad18/Rad5/Mms2 and Mgs1 are required to promote replication when forks are destabilized or stalled due to defects in Pol delta. These data are consistent with the biochemical activity of the human Mgs1 orthologue, which binds and stimulates Pol deltain vitro. We also demonstrate that Mgs1 interacts physically with Pol31 in vivo. Moreover, regions I and VII of Pol31, which are specifically sensitive to high levels of Mgs1 and PCNA, could be sites of interaction.
Vijeh Motlagh
Niloofar Davoodi
ND
Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba 6-3, Sendai, Miyagi 980-8578, Japan.
Seki
Masayuki
M
Branzei
Dana
D
Enomoto
Takemi
T
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000483:Alleles; D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003080:Cold Temperature; D017124:Conserved Sequence; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004258:DNA Polymerase III / Q000737:chemistry / Q000235:genetics; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017353:Gene Deletion; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018809:Proliferating Cell Nuclear Antigen; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013696:Temperature; D044767:Ubiquitin-Protein Ligases
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C112520:MMS2 protein, S cerevisiae; C441579:MRC1 protein, S cerevisiae; D018809:Proliferating Cell Nuclear Antigen; C063607:RAD18 protein, S cerevisiae; C088385:RAD24 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D044767:Ubiquitin-Protein Ligases; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; C095943:POL31 protein, S cerevisiae; D000251:Adenosine Triphosphatases; C506739:MGS1 protein, S cerevisiae; C076225:RAD5 protein, S cerevisiae; D004265:DNA Helicases