Human Tim-Tipin complex affects the biochemical properties of the replicative DNA helicase and DNA polymerases

Abstract

Tim (Timeless) and Tipin (Tim-interacting protein) form a stable heterodimeric complex that influences checkpoint responses and replication fork progression. We report that the Tim-Tipin complex interacts with essential replication fork proteins and affects their biochemical properties. The Tim-Tipin complex, reconstituted and purified using the baculovirus expression system, interacts directly with Mcm complexes and inhibits the single-stranded DNA-dependent ATPase activities of the Mcm2-7 and Mcm4/6/7 complexes, the DNA unwinding activity of the Mcm4/6/7 complex, and the DNA unwinding and ATPase activity of Cdc45-Mcm2-7-GINS complex, the presumed replicative DNA helicase in eukaryotes. Although stable interactions between Tim-Tipin and DNA polymerases (pols) were not observed in immunoprecipitation experiments with purified proteins, Tim was shown to interact with DNA pols α, δ, and e in cells. Furthermore, the Tim-Tipin complex significantly stimulated the pol activities of DNA pols α, δ, and e in vitro. The effects of Tim-Tipin on the catalytic activities of the Mcm complexes and DNA pols are mediated by the Tim protein alone, and distinct regions of the Tim protein are responsible for the inhibition of Mcm complex activities and stimulation of DNA pols. These results suggest that the Tim-Tipin complex might play a role in coupling DNA unwinding and DNA synthesis by directly affecting the catalytic activities of replication fork proteins.replication checkpoint | fork movement | genome integrity

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

“…The third member of the complex, called Mrc1 and claspin in budding and fission yeast and mammalian cells, respectively, is dispensable for maintenance of programmed replication fork arrest (PFA) (22,23). The FPC binds to mini chromosome maintenance (Mcm)2-7 and inhibits its DNA-dependent ATPase activity and also the helicase activity of the CMG complex (27). We have previously reported that Tof1 and Csm3 promote stable fork arrest by antagonizing the Rrm3 helicase (23,28), which removes nonhistone protein…”

The Deubiquitinase USP9X Maintains DNA Replication Fork Stability and DNA Damage Checkpoint Responses by Regulating CLASPIN during S-Phase

“…Importantly, cellular surveillance mechanisms that overlap with the DNA damage response pathways detect, stabilize, and resolve stalled replication forks to help preserve genome stability (2). CLASPIN, critical for both DNA synthesis and for signaling the presence of replication stress, is a ring-shaped protein that, together with the TIPIN-TIM1 complex, physically links DNA polymerases and DNA helicase (4)(5)(6); this is important for stabilization of replication forks, both during normal replication and upon prolonged arrest (1,2,7). CLASPIN also plays an important role in the replication stress response pathway (3,8,9), as ATR activation of CHK1 is favored by the binding of CHK1 to CLASPIN (2,(10)(11)(12)(13).…”

“…Several kinases have been reported to phosphorylate CLASPIN and promote CHK1 activation, including CHK1 itself (4)(5)(6)14), casein kinase 1 gamma 1 (CK1g1; ref. 15), and CDC7 (16,17).…”

Yeast DNA Polymerase ϵ Catalytic Core and Holoenzyme Have Comparable Catalytic Rates

“…This is very slow, considering that estimates suggest that entire Okazaki fragments are synthesized and processed in under 1 s (57, 58). The loading of Pol ⑀ might be faster in vivo due to the activities of other proteins, such as GINS in the preloading complex/CMG helicase in yeast and TIM-Tipin in human cells (25,59,60). Recent in vitro reconstitution experiments showed that CMG helicase selects Pol ⑀ over Pol ␦ on the leading strand in yeast (61).…”