A significant issue that has limited the

A significant issue that has limited the biochemical characterization of the E1 helicase to only a few PV types has been the difficulty in expressing and purifying large quantities of the protein in a recombinant form that can support cell-free DNA replication. This is in part due to the fact that E1 must assemble from monomers at the ori in order to be active for DNA replication but is typically purified as large pre-formed oligomers when overexpressed in heterologous systems such as bacteria and insect cells. A notable exception has been the BPV1 E1 protein which can be readily purified from bacteria or insect melanocortin receptor in monomeric form or as oligomers that are in a monomer–hexamer equilibrium; a feature that has contributed to making BPV1 E1 the preferred enzyme for biochemical studies (Bonne-Andrea et al., 1995a, Fouts et al., 1999, Melendy et al., 1995, Mohr et al., 1990, Müller et al., 1994, Sedman and Stenlund, 1998). In contrast and as mentioned above, overexpression of E1 from other PV types often results in enzyme preparations comprised mostly of oligomers rather than monomers. For example, HPV11 E1 overproduced in insect cells using a baculovirus-expression system is purified mostly as hexamers that are not easily dissociated into monomers and thus poorly active in cell-free DNA replication, despite displaying significant levels of ATPase and short-duplex DNA unwinding activities (Dixon et al., 2000, Rocque et al., 2000, White et al., 2001). Fortunately, this issue has not been insurmountable and protocols have been developed to obtain HPV11 E1 from insect cells in a replication-competent form (Kuo et al., 1994). Monomeric and active HPV11 E1 can also be obtained by expression of the protein in vitro, by coupled transcription and translation in a rabbit reticulocyte lysate, a facile approach that should be easily adaptable to the E1 proteins from other PV types (Amin et al., 2000). E1 is encoded by the largest and most conserved open-reading frame (ORF) of the PV genome. The protein ranges in size from 600 to 650 amino acids, depending on the PV type. Overall, the protein can be divided into three functional segments: an N-terminal regulatory region that is essential for optimal replication in vivo but dispensable in vitro (Amin et al., 2000, Ferran and McBride, 1998, Morin et al., 2011, Sun et al., 1998), a central origin-binding domain (known as the DNA-binding domain, DBD) that recognizes specific sites in the ori (Auster and Joshua-Tor, 2004, Chen and Stenlund, 1998, Leng et al., 1997, Sarafi and McBride, 1995, Sun et al., 1998, Thorner et al., 1993, Titolo et al., 2003), and a C-terminal enzymatic domain sufficient for self-assembly into hexamers that display ATPase activity and are capable of unwinding short DNA duplexes (Fig. 1) (Castella et al., 2006b, Enemark and Joshua-Tor, 2006, Titolo et al., 2000, White et al., 2001). The DBD and C-terminal helicase domain (HD) are sufficient for ori-dependent DNA replication in vitro and form the core of the molecular motor that drives viral DNA replication (Amin et al., 2000).