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Lesions in the transcribed strand block transcription and are repaired more rapidly than lesions in the nontranscribed (coding) strand which do not block RNA polymerase (RNAP). It has been shown previously that in Escherichia coli the mfd (mutation frequency decline) gene is necessary for strand-specific repair. The mfd gene was cloned and sequenced and the Mfd protein was purified and used to reconstitute strand-specific repair in a completely defined system. The mfd gene encodes a protein of 130 kilodaltons and contains the so-called "helicase motifs," a leucine zipper motif, and regions of sequence similarity to UvrB and RecG proteins. The Mfd protein was shown to (i) displace RNAP stalled at a lesion in an adenosine triphosphate-dependent reaction, (ii) bind to the damage recognition subunit (UvrA) of the excision nuclease, and (iii) stimulate the repair of the transcribed strand only when transcription is …