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Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement
Published on: January 19, 2017
Mammalian TIMELESS and Tipin are evolutionarily conserved replication fork-associated factors.
Anthony L Gotter1, Christine Suppa, Beverly S Emanuel
1Division of Human Genetics and Molecular Biology, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA. gotter@email.chop.edu
Journal of Molecular Biology
|December 5, 2006
Summary
Mammalian TIMELESS (TIM) and its partner Tipin are crucial replisome proteins. They coordinate DNA replication with genotoxic stress responses, unlike circadian-related TIM paralogs.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- The function of mammalian TIMELESS (TIM) protein was unclear, despite its essential role in embryonic development.
- TIM shares similarity with eukaryotic proteins involved in DNA synthesis and checkpoint activation at replication forks.
Purpose of the Study:
- To investigate the biochemical and cellular function of mammalian TIM and its partner Tipin.
- To determine if mammalian TIM and Tipin function as orthologs of yeast replication fork proteins.
Main Methods:
- Assessed association of TIM and Tipin with replication fork complexes using biochemical assays.
- Utilized BrdU incorporation to identify DNA replication sites.
- Performed knock-down experiments to evaluate TIM's role in DNA replication efficiency.
- Investigated the binding of the TIM-Tipin complex to replication protein A.
- Examined Tipin's role in UV and H2O2-induced checkpoint activation.
Main Results:
- Mammalian TIM and Tipin are confirmed replisome-associated proteins, binding to replication fork complexes and localizing to replication sites.
- TIM depletion impairs DNA replication efficiency.
- The TIM-Tipin complex directly binds to replication protein A, suggesting a role in coupling replication processes.
- Tipin is involved in UV-dependent and H2O2-induced DNA damage checkpoint responses.
Conclusions:
- Mammalian TIM and Tipin are functional orthologs of yeast proteins, coordinating DNA replication with genotoxic stress responses.
- This study clarifies the role of mammalian TIM as a replication-associated protein, distinct from its circadian paralogs.
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