Yeast Rev1 is cell cycle regulated, phosphorylated in response to DNA damage and its binding to chromosomes is dependent upon MEC1.
Translesion DNA synthesis (TLS) is one of the mechanisms involved in lesion bypass during DNA replication. Three TLS polymerases (Pol) are present in the yeast Saccharomyces cerevisiae: Pol zeta, Pol eta and the product of the REV1 gene. Rev1 is considered a deoxycytidyl transferase because it almost exclusively inserts a C residue in front of the lesion. Even though REV1 is required for most of the UV-induced and spontaneous mutagenesis events, the role of Rev1 is poorly understood since its polymerase activity is often dispensable. Rev1 interacts with several TLS polymerases in mammalian cells and may act as a platform in the switching mechanism required to substitute a replicative polymerase with a TLS polymerase at the sites of DNA lesions. Here we show that yeast Rev1 is a phosphoprotein, and the level of this modification is cell cycle regulated under normal growing conditions. Rev1 is unphosphorylated in G1, starts to be modified while cells are passing S phase and it becomes hyper-phosphorylated in mitosis. Rev1 is also hyper-phosphorylated in response to a variety of DNA damaging agents, including treatment with a radiomimetic drug mostly causing double-strand breaks (DSB). By using the chromosome spreading technique we found the Rev1 is bound to chromosomes throughout the cell cycle, and its binding does not significantly increase in response to genotoxic stress. Therefore, Rev1 phosphorylation does not appear to modulate its binding to chromosomes, suggesting that such modification may influence other aspects of the TLS process. Rev1 binding under damaged and undamaged conditions, is at least partially dependent on MEC1, a gene playing a pivotal role in the DNA damage checkpoint cascade. This genetic dependency may suggest a role for MEC1 in spontaneous mutagenesis events, which require a functional REV1 gene.
Sabbioneda
Simone
S
Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano., Via Celoria 26, 20133 Milano, Italy.
Bortolomai
Ileana
I
Giannattasio
Michele
M
Plevani
Paolo
P
Muzi-Falconi
Marco
M
D015825:Chromosomes, Fungal / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D004271:DNA, Fungal / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D016193:G1 Phase / Q000235:genetics*; D047908:Intracellular Signaling Peptides and Proteins; D008938:Mitosis; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D016196:S Phase / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D000099270:Y-Family DNA Polymerases
D004271:DNA, Fungal; D004259:DNA-Directed DNA Polymerase; D047908:Intracellular Signaling Peptides and Proteins; D009713:Nucleotidyltransferases; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D029701:Saccharomyces cerevisiae Proteins; C088937:MEC1 protein, S cerevisiae; C100951:REV1 protein, S cerevisiae; D000099270:Y-Family DNA Polymerases