Budding yeast, Saccharomyces cerevisiae, has a unique cell cycle. During its S-phase, DNA replication occurs, but spindle formation and chromosome-microtubule attachment also take place. Thus, how to couple spindle formation and chromosome attachment with DNA replication during S-phase is a challenge to yeast cells. In this dissertation, we investigated the differential regulation of cyclin-dependent kinase (Cdk) activity in response to DNA replication stress. In addition, we addressed how the shorter spindle structure contributes to kinetochore-microtubule interaction. Finally, the function of a kinesin complex in tension generation across sister kinetochores and kinetochore-microtubule (KT-MT) interaction has also been investigated. Periodically regulated Cdk is required for DNA synthesis and mitosis. Hydroxyurea (HU) inhibits DNA synthesis by depleting dNTPs, the basic unit for DNA synthesis. HU treatment triggers the S-phase checkpoint, which arrests cells at S-phase, inhibits late origin firing and stabilizes replication forks. Using budding yeast as a model system, we found that Swe1, a negative regulator of Cdk, is stabilized in HU-treated cells. Interestingly, this accumulation is not dependent on S-phase checkpoint. hsl1─, hsl7─, and cdc5-2 mutants, which have defects in Swe1 degradation, show HU sensitivity because of their defects in Swe1 degradation. We further demonstrated that their HU sensitivity is not a result of DNA damage accumulation or incomplete DNA synthesis; instead the sensitivity is due to their dramatically delayed recovery from HU-induced S-phase arrest. Strikingly, our in vivo data indicate that Swe1 inhibits the kinase activity of Clb2-Cdk1, but not that of Clb5-Cdk1. Therefore, S-phase accumulated Swe1 prevents Clb2-Cdk1ľmediated mitotic activities, but has little effect on Clb5-Cdk1ľassociated S-phase progression. These results explain a long-time puzzle regarding the question why the Cdk1 negative regulator Swe1, which is present in S-phase, does not affect DNA synthesis. The kinetochore is a protein complex that assembles on centromeric DNA to mediate chromosome-microtubule interactions. Most eukaryotic cells form the spindle and establish kinetochore-microtubule interactions during mitosis, but budding yeast cells finish these processes in S-phase. It has long been noticed that the S-phase spindle in budding yeast is shorter than that in metaphase, but the biological significance of this short S-phase spindle structure remains unclear. We addressed this issue by using ask1-3, a temperature sensitive kinetochore mutant that exhibits partially elongated spindles at permissive temperature in the presence of HU, a DNA synthesis inhibitor. After exposure to, and removal of, HU, ask1-3 cells show a delayed anaphase entry. This delay depends on the spindle checkpoint, which monitors kinetochore-microtubule interaction defects. Overproduction of microtubule-associated protein Ase1 or Cin8 also induces spindle elongation in HU-arrested cells. The spindle checkpoint-dependent anaphase entry delay is also observed after ASE1 or CIN8 overexpression in HU-arrested cells. Therefore, the shorter spindle in S-phase cells is likely to facilitate proper chromosome-microtubule interactions. In budding yeast, chromosomes are attached by spindle microtubules emanating from opposite spindle poles to establish bipolar attachment during S-phase. The bipolar attachment generates tension on chromosomes and defects in tension generation activate the tension checkpoint that blocks anaphase entry. The pulling force directed to the opposite spindle poles as well as the cohesin that holds sister chromatids together provides the mechanical mechanism for tension generation. Here we present evidence indicating that Cik1/Kar3, a minus-end-directed kinesin motor complex in budding yeast, binds to kinetochore protein and promotes generation of tension on sister kinetochores. Cells lacking Cik1/Kar3 exhibit tension defects as indicated by the decreased sister centromere separation in pre-anaphase cells. Moreover, cik1─ mutant cells show reduced association of centromeric DNA with Ask1, a component of DASH complex that encircles a microtubule and mediates kinetochore-microtubule interactions. Since Kar3 has been shown to drive the poleward chromosome movement along microtubules, it is likely that Cik1/Kar3-dependent chromosome transport promotes tension generation and facilitates DASH-dependent kinetochore-microtubule interactions.