The effect of regulatory sequence elements upon the initiation of DNA replication of the minute virus of mice.
The minute virus of mice (MVM) genome is a linear single-stranded length of approximately 5000 nucleotides of DNA with unique terminal palindromic sequences at both ends. The left(3') hairpin is used to prime the initiation of DNA synthesis on parental single-strand DNA while the right (5') hairpin or stem-plus-arms structure can also prime the initiation of DNA synthesis during synthesis of dimer and higher oligomers as well as synthesis of progeny single strands. Previous studies have shown that if viral duplex DNA was input into an in vitro DNA replication system using extracts from uninfected HeLa cells, the 5' end of the molecule was able to form a hairpin and initiate DNA synthesis by DNA polymerase delta (Cossons et al. (1996), Virology 216, 258-264). In this study, the effect of the deletion of known cis-acting genetic elements upon the initiation of DNA replication was studied using a series of MVM mutants with deletions within the 5' terminal region. Mutants containing deletions of elements A (nucleotides 4489-4636), B (nucleotides 4636-4695), and either one or both of the 65-bp repeats (nucleotides 4720-4785 and 4785-4849) were used as template in the in vitro DNA replication system. When element A was deleted, the efficiency of initiation decreased significantly. Subsequent removal of element B, leaving just the two 65-bp repeats, restored levels of initiation back to those seen in the wild-type genome. In the absence of either A or B both 65-bp repeats were necessary for efficient initiation, and removal of one of these repeats caused a decrease in efficiency. Thus, element B appeared to have a negative regulatory effect (in the absence of element A), and element A appeared to have a positive regulatory effect, at least in the presence of element B. These data demonstrate, for the first time, a complex interaction between these cis-acting regulatory elements which can function as both positive or negative regulators in the initiation of MVM DNA replication.
Cossons
N
N
Lady Davis Institute for Medical Research, SMBD-Jewish General Hospital, Montreal, Quebec, Canada.
Zannis-Hadjopoulos
M
M
Tam
P
P
Astell
C R
CR
Faust
E A
EA
D000818:Animals; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008915:Minute Virus of Mice / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D014779:Virus Replication
D004279:DNA, Viral