No abstract

The MCM2-7 helicase complex is loaded on DNA replication origins during the G1 phase of the cell cycle to license the origins for replication in S phase. How the initiator primase-polymerase complex, DNA polymerase ␣ (pol ␣), is brought to the origins is still unclear. We show that And-1/Ctf4 (Chromosome transmission fidelity 4) interacts with Mcm10, which associates with MCM2-7, and with the p180 subunit of DNA pol ␣. And-1 is essential for DNA synthesis and the stability of p180 in mammalian cells. In Xenopus egg extracts And-1 is loaded on the chromatin after Mcm10, concurrently with DNA pol ␣, and is required for efficient DNA synthesis. Mcm10 is required for chromatin loading of And-1 and an antibody that disrupts the Mcm10-And-1 interaction interferes with the loading of And-1 and of pol ␣, inhibiting DNA synthesis. And-1/Ctf4 is therefore a new replication initiation factor that brings together the MCM2-7 helicase and the DNA pol ␣-primase complex, analogous to the linker between helicase and primase or helicase and polymerase that is seen in the bacterial replication machinery. The discovery also adds to the connection between replication initiation and sister chromatid cohesion.[Keywords: And-1/CTF4; DNA replication; genome stability; cell cycle; DNA polymerase] Supplemental material is available at http://www.genesdev.org.

Graphical Abstract

Elevated hyaluronan biosynthesis and matrix deposition correlates with cell proliferation and migration. We ectopically expressed three isoforms of hyaluronan synthase (HAS1, HAS2, or HAS3) in nontransformed rat 3Y1 cells and observed a de novo, massive formation of a hyaluronan matrix that resulted in a partial loss of contact-mediated inhibition of cell growth and migration. All three HAS transfectants showed an enhanced motility in scratch wound assays, and a significant increase in their confluent cell densities. In high-density cultures, the HAS transfectants had a fibroblastic cell shape and markedly formed overlapping cell layers. This phenotype was more pronounced in the HAS2 transfectants than HAS1 or HAS3 transfectants, and occurred with significant alterations in the microfilament organization and N-cadherin distribution at the cell-cell border. Inhibition of a phosphatidylinositol 3-kinase (PI3-kinase) pathway resulted in reacquisition of the normal phenotype of HAS2 transfectants, suggesting that the intracellular PI3-kinase signaling regulates diminution of contact inhibition induced by formation of the massive hyaluronan matrix. Our observations suggest that hyaluronan and its matrix can modulate contact inhibition of cell growth and migration, and provide evidence for functional differences between hyaluronan synthesized by the different HAS proteins.H yaluronan (HA) is a linear polysaccharide composed of a, and is widely present as a major component of extracellular matrix in most vertebrate tissues (1, 2). HA is synthesized by three HA synthase isoforms: HAS1, HAS2, and HAS3 (3, 4). The three HAS genes have distinct expression patterns during mouse development (5), and their products have significantly different enzymatic properties and roles in the formation of the HA matrix (6).HA synthesis and matrix formation may have a role in the development, progression and pathogenesis of cancer. For example, the rate of HA synthesis is enormously increased when oncogenic viruses transform fibroblasts (7,8), and elevated levels of HA are associated with the hyperproliferative and malignant phenotypes in melanomas and various carcinomas (9-11).Studies done with cultured cancer cells showed that overproduction of HA enhances their anchorage-independent growth, tumorigenicity, and metastatic potential (12, 13), suggesting an important role for HA in tumor growth and malignant progression. However, it was unclear whether the overproduction could induce a malignant transformation of nontransformed cells.We overexpressed the three HAS isoforms to test the ability of HA to transform nontransformed cells. We also investigated whether the three HAS isoforms are functionally distinct in nontransformed cells. Materials and MethodsCell Culture and Transfection. 3Y1 1-B6 cells were obtained from Riken Cell Bank (Tsukuba, Japan). The 3Y1 cells were cultured in DMEM containing 10% FCS and 2 mM L-glutamine (growth medium). Stable transfectants were established as previously described (6) and were routinely cultured in ...