MULTIPLE CELLULAR FACTORS BIND TO CIS-REGULATORY ELEMENTS FOUND INBOARD OF THE 5' PALINDROME OF MINUTE VIRUS OF MICE

Previous genetic analysis of the DNA replication of minute virus of mi ce (MVM) minigenomes suggested that specific elements, A (nucleotides nt 4489 to 4636) and B (nt 4636 to 4695), found inboard of the 5' pa lindrome are required for efficient MVM DNA replication (P. Tam and C. R. Astell, Virology 193:812-824, 1993). In this report, we show that two MVM RsaI restriction fragments (RsaI A nt 4431 to 4579 and RsaI B nt 4579 to 4662) are able to activate DNA replication of an MVM mi nigenome containing deletions of both elements A and B. We also show t hat sequences inboard of the right palindrome are able to activate rep lication of minigenomes containing two left termini. In order to inves tigate the importance of the RsaI fragments, we demonstrate the presen ce of a number of sequence-specific DNA-protein interactions by electr ophoretic mobility shift assays. After partial fractionation of A9 nuc lear extracts, DNase I footprinting analysis was used to determine the binding sites for MVM replication factor (MRF) B5. MRF B5 protects tw o distinct regions (sites I and II) of the RsaI B probe from DNase I d igestion. Competition electrophoretic mobility shift assays with synth etic oligonucleotides corresponding to sites I and II suggest that MRF B5 is composed of two factors, MRF B3 and MRF B4, which bind DNA inde pendently in a sequence-specific manner. It may be possible that these replication factors are proteins which are able to transactivate MVM DNA replication and hence are accessory replication factors.