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. 2014 Oct 27;42(20):12912–12927. doi: 10.1093/nar/gku960

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© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 8. — Tim-Tipin-RPA complex formation relies on ssDNA length-dependent RPA conformations. (A) Graphical scheme of the dynamic formation and disruption of the Tim-Tipin-RPA-ssDNA complex in connection with the ssDNA-length dependent conformational change of RPA. RPA shows two binding modes (8 and 30 nt binding mode), which can coexist in a dynamic equilibrium in solution (top). RPA and Tim-Tipin form a complex without and in the presence of long excess ssDNA. In the DNA-free state, the RPA conformation is fixed by Tim-Tipin into a compact mode resembling RPA's conformation in the presence of long ssDNA. The Tim-Tipin-RPA-ssDNA complex dissociates in the presence of short or substoichiometric amounts of long ssDNA, thus upon the conformational change of RPA to the 8 nt binding mode. A possible ssDNA path within our Tim-Tipin-RPA EM reconstruction is indicated as dashed line. (B) Interaction scheme of RPA and ssDNA, RPA and Tim-Tipin and Tim-Tipin and ssDNA showing the K_D determined in this study (marked by star). RPA shows a very high (nano- to subnanomolar) affinity to ssDNA in comparison to Tim-Tipin (micromolar) suggesting that Tim-Tipin binding to ssDNA might play only a minor role in the Tim-Tipin-RPA complex. (C) Hypothetical role of Tim-Tipin-RPA at the replication fork. During normal DNA replication Tim-Tipin could be bound to RPA adopting its 30 nt binding mode. After recruitment to the replication sites Tim-Tipin could get loaded onto DNA replication forks by a hand-off mechanism. During this process, RPA could perform a conformational switch adopting the 8 nt binding mode and thus releasing Tim-Tipin, which in turn is placed between the helicase and polymerase. Once placed at the fork, Tim-Tipin could then couple helicase-polymerase functions. In response to replicative stress or DNA damage accumulation of ssDNA occurs, which is covered by RPA. RPA could recruit Tim-Tipin to the accumulated ssDNA, which then becomes a part of the intra-S phase checkpoint protein complex and mediates efficient phosphorylation of Chk1 by ATR (6). Conformational change of RPA through phosphorylation (57) or interaction with other proteins could lead to Tim-Tipin release, which in turn resumes RPA-independent functions like DNA binding and helicase-polymerase coupling or associates with other forks.