A framework for open discourse on the use of CRISPR-Cas9 technology to manipulate the human genome is urgently needed

No abstract

For efficient DNA replication of papillomaviruses, only two viral-encoded proteins, El and E2, are required. Other proteins and factors are provided by the host cell. E2 is an enhancer ofboth transcription and replication and is known to help El bind cooperatively to the origin of DNA replication. El is sufficient for replication in extracts prepared from permissive ceils, but the activity is enhanced by E2. Here we show that purified El can act as an ATP-dependent DNA helicase. To measure this activity, we have used strand displacement, unwinding of topologically constrained DNA, denaturation of duplex fragments, and electron microscopy. The ability of El to unwind circular DNA is found to be independent of origin-specific viral DNA sequences under a variety of experimental conditions. In unfractionated cellular extracts, El-dependent viral DNA replication is origin-dependent, but at elevated El concentrations, replication can occur on nonorigin-containing DNA templates. This conversion from an origin-dependent replication system to a nonspecific initiator system is discussed in the context of the current understanding of the initiation of chromosomal DNA replication.Helicases are critical enzymes in the semiconservative replication of DNA (1). Although some polymerases can melt duplex DNA and progressively catalyze strand displacement ahead of the growing polynucleotide chain, this melting is usually inefficient and requires the aid of a helicase. Helicases also work in conjunction with proteins having a strong affinity for single-stranded (ss) DNA, which stabilize the melted duplex as the helicase catalyzes processive unwinding. Often a helicase is brought to the site on DNA where replication initiates by interaction with specific DNA binding proteins that preassemble at the origin of DNA replication. For Escherichia coli or its A phages, the DnaB helicase is efficiently loaded onto the respective origin sites by DnaA and DnaC (2) or the A-encoded 0 and P proteins (3). In eukaryotes, little is known as to how cellular helicases may become associated with replication complexes, although, for some of the well-studied animal viruses such as the polyomaviruses and the herpesviruses, viral-encoded helicases are also equipped to be site-specific DNA binding proteins that can recognize the start site for replication. Other auxiliary factors may, therefore, not be required for loading. The simian virus 40 large tumor antigen is such an originrecognizing helicase (4) that initiates replication by unwinding the origin site as the complex cellular polymerizing machinery assembles (5, 6). Herpes simplex virus 1 (HSV-1) encodes two proteins that can catalyze DNA strand displacement on helicase substrates, and one of these proteins binds specifically to the duplex HSV-1 origin of replication, although extensive duplex unwinding has not been detected (7, 8).We have described an in vitro replication system for bovine papilloma virus 1 (BPV-1) DNA (9, 10) that may provideThe publication costs of this article were d...

Nonhomologous DNA recombination is frequently observed in somatic cells upon the introduction of DNA into cells or in chromosomal events involving sequences already stably carried by the genome. In this report, the DNA sequences at the crossover points for excision of SV40 from chromosomes were shown to be associated with eukaryotic topoisomerase I cleavage sites in vitro. The precise location of the cleavage sites relative to the crossover points has suggested a general model for nonhomologous recombination mediated by topoisomerase I.