Tipin and Timeless form a mutually protective complex required for genotoxic stress resistance and checkpoint function

Abstract

Tipin is a mammalian protein that interacts with Timeless, which plays a role in DNA damage checkpoint responses. Here, we show that Tipin is a nuclear protein that associates with the replicative helicase and protects cells against genotoxic agents. Tipin is required for efficient cell cycle arrest in response to DNA damage, and depletion of Tipin renders cells sensitive to ionizing radiation as well as replication stress. Loss of Tipin results in spontaneous ␥-H2AX foci, a marker for DNA double-strand breaks. We find that Tipin and Timeless form a complex that maintains the level of both proteins in cells and that the loss of either one will lead to the loss of the interacting partner. This observation explains the similar checkpoint phenotypes observed in both Tipin-and Timeless-depleted cells.DNA damage ͉ S phase ͉ cell cycle control ͉ circadian rhythms

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

“…Similar to previous reports using cancer cell lines, depletion of Timeless reduced the stability of Tipin and vice versa. 3,6,9,14 When NHF1-hTERT were electroporated with siRNAs targeting Chk1, Timeless, Tipin or Claspin, the targeted protein was reduced, but the levels of the other three proteins also decreased by 40-50%. These proteins may regulate each other's expression, and indeed, Chk1 has been shown to regulate the stability of Claspin.…”

“…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. 3,6,17 Timeless, Tipin and Claspin orthologs/analogs also contribute to sister chromatid cohesion (SCC) in various organisms.…”

And‐1 coordinates with Claspin for efficient Chk1 activation in response to replication stress

“…Once activated, ATR phosphorylates Chk1 in a process involving multiple replisome proteins, including Claspin, Timeless, and Tipin. These replisome proteins act coordinately to bring Chk1 to stalled forks so that Chk1 can be activated by ATR (Kumagai & Dunphy, 2000;Chini & Chen, 2003;Chou & Elledge, 2006;UnsalKacmaz et al, 2007;Yoshizawa-Sugata & Masai, 2007;Burrows & Elledge, 2008). Although Timeless, Tipin, and Claspin are critical for Chk1 activation, the mechanism by which these replisome proteins coordinate with ATR to activate Chk1 in mammalian cells remains largely unknown.…”

Separation of intra-S checkpoint protein contributions to DNA replication fork protection and genomic stability in normal human fibroblasts

“…[31][32][33][34][35][36] TIMELESS, TIPIN and CLASPIN orthologs travel with the replication fork 26,37,38 and interact with replisome components (including MCM helicase subunits, replicative polymerases, AND1, PCNA and RPA). 15,39,40 Although TIM-TIPIN mediates ATR-CHK1 signaling in response to experimentally induced replication stress, loss of TIM-TIPIN is associated with increased ssDNA and activation of CHK1, 41 suggesting that TIMELESS and TIPIN have functions for preservation of replication fork structure that are independent of ATR-CHK1 signaling. Also, several contributions of Mrc1 (yeast analog of CLASPIN) to unperturbed DNA replication are independent of its role(s) in intra-S checkpoint regulation.…”