DNA replication is a central process in all living organisms. Polyomavirus DNA replication serves as a model system for eukaryotic DNA replication and has considerably contributed to our understanding of basic replication mechanisms. However, the details of the involved processes are still unclear, in particular regarding lagging strand synthesis. To delineate the complex mechanism of coordination of various cellular proteins binding simultaneously or consecutively to DNA to initiate replication, we investigated single-stranded DNA (ssDNA) interactions by the SV40 large T antigen (Tag). Using single molecule imaging by atomic force microscopy (AFM) combined with biochemical and spectroscopic analyses we reveal independent activity of monomeric and oligomeric Tag in high affinity binding to ssDNA. Depending on ssDNA length, we obtain dissociation constants for Tag-ssDNA interactions (KD values of 10–30 nM) that are in the same order of magnitude as ssDNA binding by human replication protein A (RPA). Furthermore, we observe the formation of RPA-Tag-ssDNA complexes containing hexameric as well as monomeric Tag forms. Importantly, our data clearly show stimulation of primase function in lagging strand Okazaki fragment synthesis by monomeric Tag whereas hexameric Tag inhibits the reaction, redefining DNA replication initiation on the lagging strand.

Different tissues express different alkaline phosphatase (AP) isoforms and HPP patients' phenotypes can greatly differ in the responsiveness between tissues 1. Remarkably, comparative studies have already shown that the predominant TNAP transcript variant in bones is the same as in brain tissues 6 , which implies a common mode of TNAP function in both tissues. TNAP's most important biochemical function in bones and teeth is providing the basis for mineralization processes. The enzyme enables hydroxyapatite crystallization in bone via catalyzing the dephosphorylation of mineralization inhibitors such as inorganic pyrophosphate (PP i) and phosphorylated osteopontin 7. In the nervous system, two predominant mechanisms, the availability of vitamin B6 and alterations in purinergic signaling 8,9 , significantly influence the outcome of the disease. In case of decreased TNAP-activity, pyridoxal-5-phosphate (PLP), which is the transportable form of vitamin B6, accumulates within the serum and cannot be redistributed into the brain without dephosphorylation via TNAP. PLP is an essential enzymatic co-factor within central neurotransmitter synthesis pathways in the brain and the lack of PLP results in neurological impairment due to limited biochemical conversion 8. Additionally, TNAP enzyme supports the development and maintenance of synapse functionality and is involved in the outgrowth and myelination of neurites 10-12. Localization of TNAP within primates' brains has been described as distinct patterns within layer 4 and 5 of the cortex 13,14 and has been detected in the retina across a number of different vertebrate species 15. In mice, TNAP localization was detected predominantly in endothelial cells, primordial germ cells, pioneer growth cones, and neural precursors 16. Akp2 (the murine version of the human ALPL gene) knockout mice display reduced serum levels of alkaline phosphatase, elevated substrate levels, impaired bone mineralization, and frequently die from epileptic seizures 17,18. Due to the early lethal phenotype in mice and the lack of supplementary in vivo studies within other vertebrates, our knowledge about TNAP's interconnected functions within bone and neuronal tissues is still scarce. Due to a number of biological properties, the zebrafish (Danio rerio) has become an important vertebrate model organism for bone 19,20 and brain research 21. Along with other advantages, including low cost and easy housing of the animals, the possibility to investigate transgenic reporter lines by non-invasive in vivo monitoring and perform large scale screenings, zebrafish offer a wide repertoire of different approaches for investigation of bone and brain development 22-24. Furthermore, basic molecular pathways for vertebrate development, like bone development, are evolutionarily conserved and share common properties with other species 25,26. Consequently, a rising number of in vivo models for musculoskeletal diseases have been established in recent years 27-29 and even the molecular background of complex diseases, such...

No abstract