Refining the domain architecture model of the replication origin firing factor Treslin/TICRR

Abstract

Faithful genome duplication requires appropriately controlled replication origin firing. The metazoan Treslin/TICRR origin firing factor and its yeast orthologue Sld3 are regulation hubs of origin firing. They share the Sld3-Treslin domain (STD) and the adjacent TopBP1/Dpb11 interaction domain (TDIN). We report a revised domain architecture model of Treslin/TICRR. Complementary protein sequence analyses uncovered Ku70-homologous lower case Greek beta-barrel folds in the Treslin/TICRR middle domain (M domain) and in Sld3. Thus, the Sld3-homologous Treslin/TICRR core comprises its three central domains, M domain, STD and TDIN. This Sld3-core is flanked by non-conserved terminal domains, the CIT (conserved in Treslins) and the C-terminus. We also identified Ku70-like lower case Greek beta-barrels in MTBP and Sld7. Our binding experiments showed that the Treslin lower case Greek beta-barrel mediates interaction with the MTBP lower case Greek beta-barrel, reminiscent of the homotypic Ku70-Ku80 dimerization. This binding mode is conserved in the Sld3-Sld7 dimer. We used Treslin/TICRR domain mutants to show that all Sld3-core domains and the non-conserved terminal domains fulfil important functions during origin firing in human cells. Thus, metazoa-specific and widely conserved molecular processes cooperate during origin firing in metazoa.

The TRESLIN-MTBP complex couples completion of DNA replication with S/G2 transition

“…This suggests that origin firing activity downstream of TRESLIN is important for its control of the cell cycle. Since TOPBP1 depletion failed to reproduce the effect of TRESLIN knockdown (Figure 3B), we depleted CDC45 instead, the major regulator of origin firing downstream of TRESLIN that also requires TRESLIN phosphorylations to be incorporated into active CMGs (Ferreira et al, 2022). CDC45 depletion caused a reduction in DNA synthesis and the cell's capacity to support origin firing, which was measured by the accumulation of RPA on chromatin (a surrogate of active forks) induced by ATR inhibition (Figure S6C) (Toledo et al, 2013).…”

“…U2OS cells harboring a doxycycline-inducible TRESLIN WT or TASA construct were a kind gift from Christopher Sansam (Department of Cell Biology, University of Oklahoma). U2OS cells stably expressing AcGFP-TRESLIN WT or TASA construct were published previously (Boos et al, 2013;Ferreira et al, 2022). U2OS cells with endogenously GFP-tagged CDC45 were a kind gift from Jiri Lukas (Novo Nordisk Foundation Center for Protein Research, University of Copenhagen).…”