CRUCIFORM DNA-BINDING PROTEIN IN HELA-CELL EXTRACTION

We have analyzed by band-shift assays HeLa cell protein-DNA interactio ns on a stable cruciform DNA molecule. The stable cruciform was formed by heteroduplexing the HindIII-SphI fragment of SV40 virus DNA that c ontains the origin of replication with a derivative mutant containing a heterologous substitution at the central inverted repeat. We have id entified a novel binding activity in HeLa cell extracts with specifici ty for the cruciform-containing DNA and no apparent sequence specifici ty. The activity is protein-dependent, void of detectable nuclease act ivity, and distinct from that reported for HMG1. A cruciform binding p rotein (CBP) with an apparent molecular weight of 66 kDa was enriched from HeLa cell extracts. In addition to the CBP, we have detected sequ ence-specific binding activities to sites proximal to the cruciform. B inding to one such site is increased in the cruciform-containing heter oduplex DNA by comparison to its linear homoduplex counterpart, sugges ting transmission of structural effects by the stem-loops to their loc al environment.