While the vast majority of cellular DNA in eukaryotes is contained in long linear strands in chromosomes, we have long recognized some exceptions like mitochondrial DNA, plasmids in yeasts, and double minutes (DMs) in cancer cells where the DNA is present in extrachromosomal circles. In addition, specialized extrachromosomal circles of DNA (eccDNA) have been noted to arise from repetitive genomic sequences like telomeric DNA or rDNA. Recently eccDNA arising from unique (nonrepetitive) DNA have been discovered in normal and malignant cells, raising interesting questions about their biogenesis, function and clinical utility. Here, we review recent results and future directions of inquiry on these new forms of eccDNA.

Histone mRNA levels are cell cycle regulated, and a major regulatory mechanism is restriction of stem-loop binding protein (SLBP) to S phase. Degradation of SLBP at the end of S phase results in cessation of histone mRNA biosynthesis, preventing accumulation of histone mRNA until SLBP is synthesized just before entry into the next S phase. Degradation of SLBP requires an SFTTP (58 to 62) and KRKL (95 to 98) sequence, which is a putative cyclin binding site. A fusion protein with the 58-amino-acid sequence of SLBP (amino acids 51 to 108) fused to glutathione S-transferase (GST) is sufficient to mimic SLBP degradation at late S phase. Using GST-SLBP fusion proteins as a substrate, we show that cyclin A/Cdk1 phosphorylates Thr61. Furthermore, knockdown of Cdk1 by RNA interference stabilizes SLBP at the end of S phase. Phosphorylation of Thr61 is necessary for subsequent phosphorylation of Thr60 by CK2 in vitro. Inhibitors of CK2 also prevent degradation of SLBP at the end of S phase. Thus, phosphorylation of Thr61 by cyclin A/Cdk1 primes phosphorylation of Thr60 by CK2 and is responsible for initiating SLBP degradation. We conclude that the increase in cyclin A/Cdk1 activity at the end of S phase triggers degradation of SLBP at S/G 2 .Progression through the cell cycle is driven by a class of protein kinases, the cyclin-dependent kinases (cdk's), which are sequentially activated as cells progress from one cell cycle stage to the next. In particular, progress into S phase requires the activation of cyclin E/Cdk2, ultimately resulting in the initiation of DNA replication, and progression through mitosis requires the activation of cyclin B/Cdk1 (cdc2), resulting in nuclear envelope breakdown and chromosome condensation (27). Cyclin A/Cdk2 activity is required for continued progression through S phase (36). The targets of the cdk's are activated or inactivated by phosphorylation. A second critical regulatory mechanism for cell cycle progression is targeted proteolysis of key protein regulators (13). These include the cyclin subunits of the cdk's, critical proteins in initiation of DNA replication, and proteins responsible for maintaining chromosome pairing (6).While much is known about the events that must occur for cells to transit from G 1 to S phase and for cells to enter and exit mitosis, much less is known about the molecular events at the end of S phase, as cells progress from S to G 2 phase. S phase is characterized by replication of the chromosomes, and at the same time DNA is replicated, histone proteins must be synthesized to provide histones to assemble the newly replicated chromatin. Histone mRNAs are cell cycle regulated, and their expression is restricted to S phase. The metazoan replication-dependent histone mRNAs are the only eukaryotic cellular mRNAs that are not polyadenylated. Instead, they end in a conserved stem-loop sequence (18). Since the replication-dependent histone genes lack introns, the only processing reaction required for histone mRNA biosynthesis is cleavage of the nascent transcript to ...

In eukaryotes, bulk histone expression occurs in the S phase of the cell cycle. This highly conserved system is crucial for genomic stability and proper gene expression. In metazoans, Stem-loop binding protein (SLBP), which binds to 3' ends of canonical histone mRNAs, is a key factor in histone biosynthesis. SLBP is mainly expressed in S phase and this is a major mechanism to limit bulk histone production to the S phase. At the end of S phase, SLBP is rapidly degraded by proteasome, depending on two phosphorylations on Thr 60 and Thr 61. Previously, we showed that SLBP fragment (aa 51-108) fused to GST, is sufficient to mimic the late S phase (S/G2) degradation of SLBP. Here, using this fusion protein as bait, we performed pull-down experiments and found that DCAF11, which is a substrate receptor of CRL4 complexes, binds to the phosphorylated SLBP fragment. We further confirmed the interaction of full-length SLBP with DCAF11 and Cul4A by co-immunoprecipitation experiments. We also showed that DCAF11 cannot bind to the Thr61/Ala mutant SLBP, which is not degraded at the end of S phase. Using ectopic expression and siRNA experiments, we demonstrated that SLBP expression is inversely correlated with DCAF11 levels, consistent with the model that DCAF11 mediates SLBP degradation. Finally, we found that ectopic expression of the S/G2 stable mutant SLBP (Thr61/Ala) is significantly more toxic to the cells, in comparison to wild type SLBP. Overall, we concluded that CRL4-DCAF11 mediates the degradation of SLBP at the end of S phase and this degradation is essential for the viability of cells.

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