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The human Tim/Tipin complex coordinates an Intra-S checkpoint response to UV that slows replication fork displacement
Keziban Unsal-Kaçmaz1, Paul D Chastain, Ping-Ping Qu
1Lineberger Comprehensive Cancer Center, CB 7295, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. wkarlk@med.unc.edu
Molecular and Cellular Biology
|February 14, 2007
Summary
The Tim-Tipin complex is crucial for the intra-S checkpoint response to UV DNA damage. Tipin mediates UV-induced slowing of DNA synthesis, while Tim maintains replication fork progression without damage.
Area of Science:
- Cellular biology
- Molecular genetics
- DNA replication
Background:
- UV radiation causes DNA damage, stalling replication forks and activating the intra-S checkpoint.
- The ATR-Chk1 pathway, involving mediators like Timeless (Tim), regulates this response.
- The Tim-Tipin complex has been identified in mammalian cells.
Purpose of the Study:
- To investigate the role of the Tim-Tipin complex in the intra-S checkpoint response to UV damage.
- To elucidate the specific functions of Tim and Tipin in DNA replication and checkpoint control.
Main Methods:
- Small interfering RNA (siRNA) for gene knockdown of Tipin.
- UVC irradiation of HeLa cells.
- Immunofluorescence analysis of spread DNA fibers.
- Analysis of replication fork progression and replicon initiation.
Main Results:
- Tipin knockdown reversed the intra-S checkpoint response to UVC and affected Tim expression.
- Tipin interacts with Replication Protein A (RPA) and RPA-coated DNA.
- UVC inhibited both replicon initiation and chain elongation.
- Tim depletion reduced fork progression in undamaged cells; Tipin depletion attenuated UV-induced inhibition of chain elongation.
Conclusions:
- The Tim-Tipin complex mediates the UV-induced intra-S checkpoint.
- Tim is essential for maintaining replication fork movement in undamaged cells.
- Tipin interacts with RPA on DNA and regulates DNA chain elongation at active forks following UV damage.
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