The Human Tim/Tipin Complex Coordinates an Intra-S Checkpoint Response to UV That Slows Replication Fork Displacement

Abstract

UV-induced DNA damage stalls DNA replication forks and activates the intra-S checkpoint to inhibit replicon initiation. In response to stalled replication forks, ATR phosphorylates and activates the transducer kinase Chk1 through interactions with the mediator proteins TopBP1, Claspin, and Timeless (Tim). Murine Tim recently was shown to form a complex with Tim-interacting protein (Tipin), and a similar complex was shown to exist in human cells. Knockdown of Tipin using small interfering RNA reduced the expression of Tim and reversed the intra-S checkpoint response to UVC. Tipin interacted with replication protein A (RPA) and RPA-coated DNA, and RPA promoted the loading of Tipin onto RPA-free DNA. Immunofluorescence analysis of spread DNA fibers showed that treating HeLa cells with 2.5 J/m2 UVC not only inhibited the initiation of new replicons but also reduced the rate of chain elongation at active replication forks. The depletion of Tim and Tipin reversed the UV-induced inhibition of replicon initiation but affected the rate of DNA synthesis at replication forks in different ways. In undamaged cells depleted of Tim, the apparent rate of replication fork progression was 52% of the control. In contrast, Tipin depletion had little or no effect on fork progression in unirradiated cells but significantly attenuated the UV-induced inhibition of DNA chain elongation. Together, these findings indicate that the Tim-Tipin complex mediates the UV-induced intra-S checkpoint, Tim is needed to maintain DNA replication fork movement in the absence of damage, Tipin interacts with RPA on DNA and, in UV-damaged cells, Tipin slows DNA chain elongation in active replicons.

Protumorigenic role of Timeless in hepatocellular carcinoma

“…In addition, phylogenetic sequence analysis revealed the presence of a paralogue in D. melanogaster (4), which is not involved in the core clock machinery, but is important for the maintenance of chromosome integrity, growth control, and development. In contrast, a single TIM gene has been identified in mammals, which acquired all of these functions as indicated by its role in DNA damage response, replication, and circadian rhythm (5)(6)(7)(8)(9). Consistently, TIM knockout resulted in embryonic lethality in mice (10).…”

Regulation of human DNA polymerase delta in the cellular responses to DNA damage

“…Inhibition of global DNA synthesis can result from the inhibition of the firing of new origins at low-UV doses as well as the inhibition of DNA elongation, which occurs at higher UV doses [Kaufmann, 2010]. Direct assessment of replication fork progression using combed DNA and dual-pulse labeling with halogenated nucleotides has confirmed that the progression of replication forks has slowed down, that is, rates of chain elongation are reduced [Unsal-Kacmaz et al, 2007]. Similar effects have been observed when camptothecin is used to activate the intra-S-phase checkpoint [Chastain et al, 2006;Conti et al, 2007].…”

Timeless functions independently of the Tim-Tipin complex to promote sister chromatid cohesion in normal human fibroblasts

“…9,10 SiRNA-mediated reduction of Timeless, Tipin or Claspin proteins attenuates DNA damageinduced phosphorylation of Chk1 and compromises ultraviolet (UV) light-induced activation of the intra-S checkpoint in HeLa cells. 3,6,8,11 Tim-Tipin and Claspin likely work together to mediate ATR activation of Chk1 by Tipin-RPA recruitment of ClaspinChk1 through a Tipin-Claspin interaction. 8 Furthermore, TimTipin and Claspin orthologs/analogs associate with chromatin, interact with replisome components and appear to travel with replication forks in the absence of exogenously applied DNA damage, 9,10,[12][13][14][15][16] and DNA synthesis is compromised when human cells are depleted of Timeless, Tipin or Claspin.…”

“…8 Furthermore, TimTipin and Claspin orthologs/analogs associate with chromatin, interact with replisome components and appear to travel with replication forks in the absence of exogenously applied DNA damage, 9,10,[12][13][14][15][16] and DNA synthesis is compromised when human cells are depleted of Timeless, Tipin or Claspin. 3,6,17 Timeless, Tipin and Claspin orthologs/analogs also contribute to sister chromatid cohesion (SCC) in various organisms. 9,[18][19][20][21][22] SCC is required for identical partitioning of duplicated genomes to daughter cells.…”

“…1 Timeless, Tipin and Claspin are mediators of ATR phosphorylation of Chk1 in response to DNA damage and replication stress. [3][4][5][6][7][8] Timeless and Tipin form a heterodimer and are components of the "replication fork protection complex" (RFPC), named for the timeless-tipin complex and Claspin are mediators of the AtR-dependent activation of Chk1 in the intra-S checkpoint response to stalled DNA replication forks. tim-tipin and Claspin also contribute to sister chromatid cohesion (SCC) in various organisms, likely through a replication-coupled process.…”