The Human Tim-Tipin Complex Interacts Directly with DNA Polymerase ϵ and Stimulates Its Synthetic Activity

• Published in: The Journal of biological chemistry 2013-05, Vol.288 (18), p.12742-12752
• Main Authors: Aria, Valentina, De Felice, Mariarita, Di Perna, Roberta, Uno, Shuji, Masai, Hisao, Syväoja, Juhani E., van Loon, Barbara, Hübscher, Ulrich, Pisani, Francesca M.
• Format: Article
• Language: English
• Subjects: Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cell Cycle Proteins; Cell Line; DNA - biosynthesis; DNA - chemistry; DNA - genetics; DNA and Chromosomes; DNA Damage; DNA Enzymes; DNA Polymerase; DNA Polymerase II - chemistry; DNA Polymerase II - genetics; DNA Polymerase II - metabolism; DNA Repair; DNA Replication; DNA-Binding Proteins; DNA-Protein Interaction; Fork Protection Complex; Humans; Multiprotein Complexes - chemistry; Multiprotein Complexes - genetics; Multiprotein Complexes - metabolism; Nuclear Proteins - chemistry; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Surface Plasmon Resonance - methods; Tim-Tipin Complex
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.398073

The Tim-Tipin complex plays an important role in the S phase checkpoint and replication fork stability in metazoans, but the molecular mechanism underlying its biological function is poorly understood. Here, we present evidence that the recombinant human Tim-Tipin complex (and Tim alone) markedly enhances the synthetic activity of DNA polymerase ϵ. In contrast, no significant effect on the synthetic ability of human DNA polymerase α and δ by Tim-Tipin was observed. Surface plasmon resonance measurements and co-immunoprecipitation experiments revealed that recombinant DNA polymerase ϵ directly interacts with either Tim or Tipin. In addition, the results of DNA band shift assays suggest that the Tim-Tipin complex (or Tim alone) is able to associate with DNA polymerase ϵ bound to a 40-/80-mer DNA ligand. Our results are discussed in view of the molecular dynamics at the human DNA replication fork. Background: The Tim-Tipin complex plays a critical role in the S phase checkpoint and replication fork stability by a molecular mechanism not yet elucidated. Results: The human Tim-Tipin complex specifically enhances the synthetic activity of DNA polymerase ϵ. Conclusion: The Tim-Tipin complex could modulate the DNA polymerase ϵ function at the replication fork. Significance: These findings further our understanding of the replication fork dynamics in metazoans.