SCF(Dia2) regulates DNA replication forks during S-phase in budding yeast.
Dia2 is an F-box protein, which is involved in the regulation of DNA replication in the budding yeast Saccharomyces cerevisiae. The function of Dia2, however, remains largely unknown. In this study, we report that Dia2 is associated with the replication fork and regulates replication fork progression. Using modified yeast two-hybrid screening, we have identified components of the replisome (Mrc1, Ctf4 and Mcm2), as Dia2-binding proteins. Mrc1 and Ctf4 were ubiquitinated by SCF(Dia2) both in vivo and in vitro. Domain analysis of Dia2 revealed that the leucine-rich repeat motif was indispensable for the regulation of replisome progression, whereas the tetratricopeptide repeat (TPR) motif was involved in the interaction with replisome components. In addition, the TPR motif was shown to be involved in Dia2 stability; deleting the TPR stabilized Dia2, mimicking the effect of DNA damage. ChIP-on-chip analysis illustrated that Dia2 localizes to the replication fork and regulates fork progression on hydroxyurea treatment. These results demonstrate that Dia2 is involved in the regulation of replisome activity through a direct interaction with replisome components.
Mimura
Satoru
S
Division of Biological Science, Graduate school of Science, Nagoya University, Chikusa-ku, Nagoya, Japan.
Komata
Makiko
M
Kishi
Tsutomu
T
Shirahige
Katsuhiko
K
Kamura
Takumi
T
D020816:Amino Acid Motifs / Q000502:physiology; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000378:metabolism; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D044783:F-Box Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D007930:Leucine / Q000378:metabolism / Q000502:physiology; D055550:Protein Stability; D017434:Protein Structure, Tertiary / Q000502:physiology; D020449:Repetitive Sequences, Amino Acid / Q000502:physiology; D016196:S Phase / Q000235:genetics / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*
C078338:CTF4 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; C495983:Dia2 protein, S cerevisiae; D044783:F-Box Proteins; C441579:MRC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C069199:MCM2 protein, S cerevisiae; D007930:Leucine