Human Timeless and Tipin stabilize replication forks and facilitate sister-chromatid cohesion

Abstract

SummaryThe Timeless-Tipin protein complex has been reported to be important for replication checkpoint and normal DNA replication processes. However, the precise mechanisms by which Timeless-Tipin preserves genomic integrity are largely unclear. Here, we describe the roles of Timeless-Tipin in replication fork stabilization and sister chromatid cohesion. We show in human cells that Timeless is recruited to replication origin regions and dissociate from them as replication proceeds. Cdc45, which is known to be required for replication fork progression, shows similar patterns of origin association to those of Timeless. Depletion of Timeless-Tipin causes chromosome fragmentation and defects in damage repair in response to fork collapse, suggesting that it is required for replication fork maintenance under stress. We also demonstrate that depletion of Timeless-Tipin impairs sister chromatid cohesion and causes a defect in mitotic progression. Consistently, Timeless-Tipin co-purifies with cohesin subunits and is required for their stable association with chromatin during S phase. Timeless associates with the cohesion-promoting DNA helicase ChlR1, which, when overexpressed, partially alleviates the cohesion defect of cells depleted of Timeless-Tipin. These results suggest that Timeless-Tipin functions as a replication fork stabilizer that couples DNA replication with sister chromatid cohesion established at replication forks.

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.…”

“…6,9,14 Leman et al (2010) 9 found that Timeless interacted with cohesin ring subunits in the presence or absence of DNA, but that the interaction of Tipin with cohesins was DNAdependent, suggestive of different contributions to SCC from Timeless versus Tipin. However, depletion of Timeless or Tipin produced equivalent (four-fold) increases in discohesion in HeLa cells.…”

“…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.…”

“…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.…”

“…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. The cohesin complex, a multi-subunit ring, creates physical linkages between sister chromatids.…”