Replication initiator protein NS1 of the parvovirus minute virus of mice binds to modular divergent sites distributed throughout duplex viral DNA.
To initiate DNA synthesis, the NS1 protein of minute virus of mice (MVM) first binds to a simple cognate recognition sequence in the viral origins, comprising two to three tandem copies of the tetranucleotide TGGT. However, this motif is also widely dispersed throughout the viral genome. Using an immunoselection procedure, we show that NS1 specifically binds to many internal sites, so that all viral fragments of more than approximately 170 nucleotides effectively compete for NS1, often binding with higher affinity to these internal sites than to sites in the origins. We explore the diversity of the internal sites using competitive binding and DNase I protection assays and show that they vary between two extreme forms. Simple sites with three somewhat degenerate, tandem TGGT reiterations bind effectively but are minimally responsive to ATP, while complex sites, containing multiple variably spaced TGGT elements arranged as opposing clusters, bind NS1 with an affinity that can be enhanced approximately 10-fold by ATP. Using immuno-selection procedures with randomized sequences embedded within specific regions of the genome, we explore possible binding configurations in these two types of site. We conclude that binding is modular, combinatorial, and highly flexible. NS1 recognizes two to six variably spaced, more-or-less degenerate forms of the 5'-TGGT-3' motif, so that it binds efficiently to a wide variety of sequences. Thus, despite complex coding constraints, binding sites are configured at frequent intervals throughout duplex forms of viral DNA, suggesting that NS1 may serve as a form of chromatin to protect and tailor the environment of replicating genomes.
Cotmore
Susan F
SF
Department of Laboratory Medicine, Yale University Medical School, 333 Cedar Street, New Haven, CT 06510, USA.
Gottlieb
Robert L
RL
Tattersall
Peter
P
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D018983:DNA Footprinting; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008915:Minute Virus of Mice / Q000502:physiology*; D011485:Protein Binding; D017361:Viral Nonstructural Proteins / Q000378:metabolism*
D004279:DNA, Viral; C081367:NS1 protein, minute virus of mice; D017361:Viral Nonstructural Proteins; D000255:Adenosine Triphosphate