It has been thought that clathrin-mediated endocytosis is regulated by phosphorylation and dephosphorylation of many endocytic proteins, including amphiphysin I and dynamin I. Here, we show that Cdk5/p35-dependent cophosphorylation of amphiphysin I and dynamin I plays a critical role in such processes. Cdk5 inhibitors enhanced the electric stimulation–induced endocytosis in hippocampal neurons, and the endocytosis was also enhanced in the neurons of p35-deficient mice. Cdk5 phosphorylated the proline-rich domain of both amphiphysin I and dynamin I in vitro and in vivo. Cdk5-dependent phosphorylation of amphiphysin I inhibited the association with β-adaptin. Furthermore, the phosphorylation of dynamin I blocked its binding to amphiphysin I. The phosphorylation of each protein reduced the copolymerization into a ring formation in a cell-free system. Moreover, the phosphorylation of both proteins completely disrupted the copolymerization into a ring formation. Finally, phosphorylation of both proteins was undetectable in p35-deficient mice.

Topoisomerase inhibitors such as camptothecin and etoposide are used as anti-cancer drugs and induce double-strand breaks (DSBs) in genomic DNA in cycling cells. These DSBs are often covalently bound with polypeptides at the 3′ and 5′ ends. Such modifications must be eliminated before DSB repair can take place, but it remains elusive which nucleases are involved in this process. Previous studies show that CtIP plays a critical role in the generation of 3′ single-strand overhang at “clean” DSBs, thus initiating homologous recombination (HR)–dependent DSB repair. To analyze the function of CtIP in detail, we conditionally disrupted the CtIP gene in the chicken DT40 cell line. We found that CtIP is essential for cellular proliferation as well as for the formation of 3′ single-strand overhang, similar to what is observed in DT40 cells deficient in the Mre11/Rad50/Nbs1 complex. We also generated DT40 cell line harboring CtIP with an alanine substitution at residue Ser332, which is required for interaction with BRCA1. Although the resulting CtIPS332A/−/− cells exhibited accumulation of RPA and Rad51 upon DNA damage, and were proficient in HR, they showed a marked hypersensitivity to camptothecin and etoposide in comparison with CtIP+/−/− cells. Finally, CtIPS332A/−/−BRCA1−/− and CtIP+/−/−BRCA1−/− showed similar sensitivities to these reagents. Taken together, our data indicate that, in addition to its function in HR, CtIP plays a role in cellular tolerance to topoisomerase inhibitors. We propose that the BRCA1-CtIP complex plays a role in the nuclease-mediated elimination of oligonucleotides covalently bound to polypeptides from DSBs, thereby facilitating subsequent DSB repair.

Two isoforms of DNA topoisomerase II (topo II) have been identified in mammalian cells. While topo II␣ is essential for chromosome segregation in mitotic cells, in vivo function of topo II␤ remains to be clarified. Here we demonstrate that the nucleoplasmic topo II␤, highly expressed in differentiating cerebellar neurons, is the catalytically competent entity operating directly on chromatin DNA in vivo. When the cells reached terminal differentiation, this in vivo activity decreased to a negligible level with concomitant loss of the nucleoplasmic enzyme. Effects of topo II-specific inhibitors were analyzed in a primary culture of cerebellar granule neurons that can mimic the in vivo situation. Only the ␤ isoform was expressed in granule cells differentiating in vitro. ICRF-193, a catalytic topo II inhibitor, suppressed the transcriptional induction of amphiphysin I which is essential for mature neuronal activity. The effect decreased significantly as the cells differentiate. Expression profiling with a cDNA macroarray showed that 18% of detectable transcripts were up-regulated during the differentiation and one-third of them were susceptible to ICRF-193. The results suggest that topo II␤ is involved in an early stage of granule cell differentiation by potentiating inducible neuronal genes to become transcribable probably through alterations in higher order chromatin structure. DNA topoisomerase II (topo II)1 is essential for cell proliferation since some mitotic events such as condensation and segregation of daughter chromosomes are entirely dependent on its activity. It is also likely to be involved in other DNA transactions like DNA replication, transcription, and recombination (1). The enzyme, in a heart-shaped dimer form (2), catalyzes topological transformations of DNA by transient cleavage of DNA double strands forming an enzyme-operated gate, followed by passing a second double-stranded DNA segment through the gate (3, 4). Only one form of topo II is present in lower eukaryotes and invertebrates whereas in mammalian cells two isoforms, the 170-kDa topo II␣ and the 180-kDa topo II␤, that are encoded by separate genes have been identified (5-7). These isoforms may share their roles in different cellular functions although they show similar catalytic properties in vitro. From the following observations, topo II␣ is considered to be the main isoform involved in the mitotic processes. First, there is a positive correlation between the cellular concentration of topo II␣ and the rate of cell proliferation (8, 9). Second, the expression of mRNA for topo II␣ is higher in tissues containing proliferating cells (10) and is localized in brain regions enriched with mitotic cells (11,12). Third, the level of topo II␣ protein peaks at G 2 /M phase during the cell cycle (9, 13) and finally, high affinity binding of topo II␣ with chromatin at mitosis is essential for chromosome condensation/segregation and topo II␤ cannot be substituted for topo II␣ (14).In contrast to topo II␣, functional aspect of topo II␤ remains obscu...

DNA topoisomerase II (topo II) catalyzes a strand passage reaction in that one duplex is passed through a transient brake or gate in another. Completion of late stages of neuronal development depends on the presence of active β isoform (topo IIβ). The enzyme appears to aid the transcriptional induction of a limited number of genes essential for neuronal maturation. However, this selectivity and underlying molecular mechanism remains unknown. Here we show a strong correlation between the genomic location of topo IIβ action sites and the genes it regulates. These genes, termed group A1, are functionally biased towards membrane proteins with ion channel, transporter, or receptor activities. Significant proportions of them encode long transcripts and are juxtaposed to a long AT-rich intergenic region (termed LAIR). We mapped genomic sites directly targeted by topo IIβ using a functional immunoprecipitation strategy. These sites can be classified into two distinct classes with discrete local GC contents. One of the classes, termed c2, appears to involve a strand passage event between distant segments of genomic DNA. The c2 sites are concentrated both in A1 gene boundaries and the adjacent LAIR, suggesting a direct link between the action sites and the transcriptional activation. A higher-order chromatin structure associated with AT richness and gene poorness is likely to serve as a silencer of gene expression, which is abrogated by topo IIβ releasing nearby genes from repression. Positioning of these genes and their control machinery may have developed recently in vertebrate evolution to support higher functions of central nervous system.