NUCLEOTIDE-SEQUENCE AND ROLE IN DNA AMPLIFICATION OF THE DIRECT REPEATS COMPOSING THE AMPLIFIABLE ELEMENT AUD1 OF STREPTOMYCES-LIVIDANS 66

The amplifiable unit of DNA no. 1 (AUD1) of Streptomyces lividans cons ists of three 1 kb repeats (left direct repeat, LDR; middle direct rep eat, MDR; and the slightly different right direct repeat, RDR) and two 4.7 kb repeats alternately arranged in identical orientation to each other, Both 4.7 kb repeats have been sequenced, They are identical and contain one open reading frame (orf4.7), The deduced amino acid seque nce has abind to an imperfect palindromic sequence upstream from MDR a nd RDR and to another sequence downstream from RDR, An extrachromosoma l DNA amplification system was constructed containing different combin ations of the sequences composing AUD1, In mutants having a deletion o f the chromosomal AUD1, the 4,7 kb repeats could be reduced in size, m utated or replaced by E. coli DNA without altering the ability to ampl ify when RDR was present, Therefore, the only function of the 4.7 kb r epeats in amplification is to provide directly repeated DNA sequences, When RDR was lacking or mutated, no amplification was observed, This strongly suggests low similarity to chitinases, and two amino acid rep eats present high similarities to fibronectin type III modules, Sequen cing had previously shown that the ORF corresponding to each 1 kb repe at encodes a putative DNA-binding protein, Crude extracts of Escherich ia coli overexpressing the orfRDR-encoded protein and of S. lividans J ni1, having a high amplification of AUD1 and therefore orfMDR, were us ed in gel retardation assays, The orfRDR- and probably the orfMDR-enco ded proteins can that the DNA-binding protein encoded by orfRDR is req uired for AUD1 amplification.