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.

The eukaryotic cell replicates its chromosomal DNA with almost absolute fidelity in the course of every cell cycle. This accomplishment is remarkable considering that the conditions for DNA replication are rarely ideal. The replication machinery encounters a variety of obstacles on the chromosome, including damaged template DNA. In addition, a number of chromosome regions are considered to be difficult to replicate owing to DNA secondary structures and DNA binding proteins required for various transactions on the chromosome. Under these conditions, replication forks stall or break, posing grave threats to genomic integrity. How does the cell combat such stressful conditions during DNA replication? The replication fork protection complex (FPC) may help answer this question. Recent studies have demonstrated that the FPC is required for the smooth passage of replication forks at difficult-to-replicate genomic regions and plays a critical role in coordinating multiple genome maintenance processes at the replication fork.

The pathogenic yeast Cryptococcus neoformans causes fungal meningitis in immune-compromised patients. Cell proliferation in the budding yeast form is required for C. neoformans to infect human hosts, and virulence factors such as capsule formation and melanin production are affected by cell-cycle perturbation. Thus, understanding cell-cycle regulation is critical for a full understanding of virulence factors for disease. Our group and others have demonstrated that a large fraction of genes in Saccharomyces cerevisiae is expressed periodically during the cell cycle, and that proper regulation of this transcriptional program is important for proper cell division. Despite the evolutionary divergence of the two budding yeasts, we found that a similar percentage of all genes (~20%) is periodically expressed during the cell cycle in both yeasts. However, the temporal ordering of periodic expression has diverged for some orthologous cell-cycle genes, especially those related to bud emergence and bud growth. Genes regulating DNA replication and mitosis exhibited a conserved ordering in both yeasts, suggesting that essential cell-cycle processes are conserved in periodicity and in timing of expression (i.e. duplication before division). In S. cerevisiae cells, we have proposed that an interconnected network of periodic transcription factors (TFs) controls the bulk of the cell-cycle transcriptional program. We found that temporal ordering of orthologous network TFs was not always maintained; however, the TF network topology at cell-cycle commitment appears to be conserved in C. neoformans. During the C. neoformans cell cycle, DNA replication genes, mitosis genes, and 40 genes involved in virulence are periodically expressed. Future work toward understanding the gene regulatory network that controls cell-cycle genes is critical for developing novel antifungals to inhibit pathogen proliferation.

The blood stage of the infection of the malaria parasite Plasmodium falciparum exhibits a 48-hour developmental cycle that culminates in the synchronous release of parasites from red blood cells, which triggers 48-hour fever cycles in the host. This cycle could be driven extrinsically by host circadian processes or by a parasite-intrinsic oscillator. To distinguish between these hypotheses, we examine the P. falciparum cycle in an in vitro culture system and show that the parasite has molecular signatures associated with circadian and cell cycle oscillators. Each of the four strains examined has a different period, which indicates strain-intrinsic period control. Finally, we demonstrate that parasites have low cell-to-cell variance in cycle period, on par with a circadian oscillator. We conclude that an intrinsic oscillator maintains Plasmodium’s rhythmic life cycle.