The 0 degree C closed complexes between Escherichia coli RNA polymerase and two promoters, T7-A3 and lacUV5

The promoter-specific binding of Escherichia coli RNA polymerase to the T7-A3 and the lacUV5 promoters at 0 degrees C was analyzed by DNase I footprinting. At 37 degrees C, the footprint from RNA polymerase bound to the A3 promoter is essentially the same as that reported by Galas, D.J., and Schmitz, A., (1978) Nucleic Acids Res. 5, 3157-3170 for the lacUV5 promoter. At 0 degrees C, the footprint for the A3 promoter is well defined but reduced in size. The principal difference between the 0 and 37 degrees C footprints is a region from -2 to +18 which is protected by polymerase at the higher but not at the lower temperature. In contrast, the 0 degree C footprint for the lacUV5 promoter differs substantially in character from the footprint for A3 at 0 degree C. The footprint is similar to the pattern of DNase I digestion of DNA bound to a surface; alternating regions of sensitive and protected DNA are spaced at intervals of about 10 base pairs. This region of DNase I-sensitive and -resistant DNA has the same boundaries as the 0 degree C footprint on T7-A3. Temperature shift experiments confirmed the sequence specificity of the RNA polymerase interaction with UV5 at 0 degree C. These results indicate that RNA polymerase binds specifically to each promoter sequence in a closed complex. The increased time and amounts of RNA polymerase required to form the 0 degree C footprint on the lacUV5 promoter indicate that it binds RNA polymerase more weakly than does the T7-A3 promoter. Therefore there is a correlation between the binding constant for closed complex formation estimated from kinetic measurements and the formation of the 0 degree C footprint. The -35 region of the promoter may be more important in establishing the 0 degree C footprint because the T7-A3 promoter is a better match to the consensus sequence. Conversely, the -10 region seems less important because lacUV5 is a perfect match to the consensus, whereas the T7-A3 promoter matches at only five out of seven positions. The 0 degree C footprints encompass both regions along with the spacer; the combination of these regions rather than an individual region may determine the character of the footprint and the magnitude of the binding constant.     

Footprint analysis of the bsp RI DNA methyltransferase-DNA interaction.

The interaction between the GGCC-specific Bsp RI DNA methyltransferase (M. Bsp RI) and substrate DNA was studied with footprinting techniques using a DNA fragment that was unmodified on both strands. Footprinting with DNase I revealed an approximately 14 bp protected region. Footprinting with dimethylsulfate detected major groove interactions with the guanine bases of the recognition sequence. Reaction with 1,10-phenanthroline-copper did not show protection, suggesting that minor groove interactions play little role in sequence-specific recognition by M. Bsp RI. Hydroxyl radical footprinting revealed a protected stretch of 6 nt. The hydroxyl radical footprint of M. Bsp RI differs markedly from the the footprint reported for the Hha I and Sss I methyltransferases. The pattern of protection from dimethylsulfate and hydroxyl radicals suggests that the interactions of M. Bsp RI with DNA are similar to those detected in the co-crystal structure of the Hae III methyltransferase.     

Induction of DNA replication-mediated double strand breaks by psoralen DNA interstrand cross-links

The effect of DNA interstrand cross-links (cross-links) on DNA replication was examined with a cell-free SV40 origin-dependent DNA replication system. A defined template DNA with a single psoralen cross-link and the SV40 origin of replication was replicated by HeLa cell-free extract in the presence of SV40 large T antigen. The psoralen cross-link inhibited DNA replication by terminating chain elongation at 1-50 nucleotides before the cross-linked sites. The termination of DNA replication by the cross-links mediated the generation of double strand breaks near the cross-linked sites. These results are the first biochemical evidence of the generation of double strand breaks by DNA replication.     

Topography of intermediates in transcription initiation of E.coli

Three characteristic footprinting patterns resulted from probing the Escherichia coli RNA polymerase T7 A1 promoter complex by hydroxyl radicals in the temperature range between 4 degrees C and 37 degrees C. These were attributed to the closed complex, the intermediate complex and the open complex. In the closed complex, the RNA polymerase protects the DNA only at one side over five helical turns. In the intermediate complex, the range of the protected area is extended further downstream by two helical turns. This region of the DNA helix is fully protected, indicating that the RNA polymerase wraps around the DNA between base positions -13 and +20. In the open complex, a stretch between base positions -7 and +2, which was fully protected in the intermediate complex, becomes accessible towards hydroxyl radicals but only in the codogenic strand, indicating that the DNA strands are unwound. Our data suggest that only the DNA downstream of the promoter is involved in this unwinding process.