Biochemical and biophysical characterization of the human TIM/TIPIN/CRY complex - a potential direct link between the circadian clock and the cell cycle
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Abstract
The circadian clock controls the behavior of many lifeforms and is functionally connected to
DNA replication and DNA damage response. Central circadian regulators influence cell cycle
and DNA damage response by altering protein expression of regulatory factors or by interacting
with them. The central circadian repressor CRY interacts with the TIM/TIPIN complex, that is
involved in signaling of stalled replication forks and DNA damage. CRY affects TIMs’ ability
to activate the CHK1 kinase which in turn leads to activation of cell cycle checkpoints and
repair of DNA damage. It is not clear how the interaction is achieved, whether there is a direct
interaction of both proteins or if they are parts of a larger complex.
With purified proteins we showed that CRY bound TIM/TIPIN in a direct manner and formed
a stoichiometric trimeric complex. We found that the two human CRY paralogs (CRY1 and
CRY2) differ in their affinity to TTP and that CRY2 bound with higher affinity. This was
possibly due to the highly variable tails between CRY1 and CRY2 as most divergent feature
between CRY1 and CRY2. We furthermore confirmed data from literature, that the TIM NTerminus
is sufficient to bind CRY. A functionally undescribed loop within TIM was
dispensable for CRY binding, but caused both CRYs to bind with different affinities, indicating
a further regulatory detail. Using Small-Angle X-Ray scattering and Crosslink MS, we created
a hypothetical model to visualize the interaction of CRY and TIM. Initial pulldown experiments
identified candidate proteins that might be affected by the TIM loop.
The observation of a direct trimeric complex of TTP/CRY indicates that CRY and TIM/TIPIN
functionally connect the circadian clock with signaling of DNA damage and replication stress.
Different binding affinities of CRY1 and CRY2 to TTP give a hint in order to understand
functional differences between the CRY paralogs and indicate functional importance of the Cterminal
CRY tails. The TIM loop was not functionally described before and its effect on CRY
binding could indicate an additional regulatory detail. The candidate proteins identified in a
TIM loop dependent pulldown could serve as a starting point for functional characterization of
the TIM loop.