The right-end hairpin of MVM contains 248 nucleotides organized into a cruciform shape. This region is almost perfectly basepaired, with just three unpaired bases at the axis and a mismatched region positioned 20 nucleotides from the axis. A three nucleotide insertion, AGA or TCT, on one strand separates opposing pairs of NS1 binding sites, creating a 36 basepair-length palindrome that can assume an alternate cruciform configuration. This configuration is expected to destabilize the duplex, which facilitates its ability to function as a hinge. The mismatch of the unpaired bases, rather than the three-nucleotide sequence itself, may help to promote instability of duplex DNA.
Fully-duplex linear forms of the right-end hairpin sequence also function as NS1-dependent origins. For many parvoviral telomeres, however, only an initiator binding site next to the nick site is required for the origin function so that the minimal sequences required for nicking are less than 40 basepairs in length. For MVM, the minimal right-end origin is around 125 basepairs in length and includes most of the hairpin sequence because at least three recognition elements are involved: the nick site 5′-CTWWTCA-3′ (element 1), positioned seven nucleotides upstream from a duplex NS1-binding site (element 2) that is oriented to have the attached NS1 complex extending over the nick site, and a second NS1-binding site (element 3), which is adjacent to the hairpin axis.
The second binding site is over 100 basepairs away from the nick site but is required for NS1-mediated cleavage. In vivo, there is slight variation in the position of the nick, plus or minus one nucleotide, with one position preferred. During nicking, this site is likely exposed as a single strand and is potentially stabilized as a minimal stem-loop by the tetranucleotide inverted repeats to the sides of the site. Optimal forms of the NS1-binding site contain at least three tandem copies of the 5′-ACCA-3′ sequence. Modest alterations to these motifs only have a small effect on affinity, which suggests that each tetranucleotide motif is recognized by different molecules in the NS1 complex. The NS1-binding site that positions NS1 over the nick site in the right-end origin is a high affinity site.
With ATP, NS1 binds asymmetrically over the aforementioned sequence, protecting a region 41 basepairs in length from digestion. This footprint extends just five nucleotides beyond the 3′-end of the ACCA repeat but 22 nucleotides beyond the 5′-end so that the footprint ends 15 nucleotides beyond the nick site, placing NS1 in position to nick the origin. Nicking only occurs if the second, distant NS1-binding site is also present in the origin and the entire complex is activated by addition of HMG1.
In the absence of NS1, HMG1 binds the hairpin sequence independently, causing it to bend, without protecting any region from digestion. HMG1 can also directly bind to NS1 and mediates interactions between NS1 molecules bound to their recognition elements in the origin, so it is essential for formation of the cleavage complex. The ability of the axis region to reconfigure into a cruciform does not appear to be important in this process. Cleavage is dependent on the correct spacing of the elements of the origin, so additions and deletions can be lethal, whereas substitutions can be tolerated. Addition of HMG1 appears to only slightly adjust the sequences protected by NS1, but the conformation of the intervening DNA changes, folding into a double helical loop that extends about 30 basepairs through a guanine-rich element in the hairpin stem. Between this element and the nick site there are five thymidine residues included in the loop, and the site has a region to its side containing many alternating adenine and thymine residues, which likely increases flexibility. The creation of the loop likely allows the terminus to assume a specific 3-dimensional structure required to activate the nickase since origins that fail to reconfigure into a double-helical loop once HMG1 is added are not nicked.