Unfolding of quadruplex structure in the G-rich strand of the minisatellite repeat by the binding protein UP1.

Abstract

The mouse hypervariable minisatellite (MN) Pc-1 consists of tandem repeats of d(GGCAG) and flanked sequences. We have previously demonstrated that single-stranded d(GGCAG)(n) folds into the intramolecular folded-back quadruplex structure under physiological conditions. Because DNA polymerase progression in vitro is blocked at the repeat, the characteristic intramolecular quadruplex structure of the repeat, at least in part, could be responsible for the hypermutable feature of Pc-1 and other MNs with similar repetitive units. On the other hand, we have isolated six MN Pc-1 binding proteins (MNBPs) from nuclear extracts of NIH 3T3 cells. Here, we describe one of those MNBPs, MNBP-B, that binds to the single-stranded d(GGCAG)(n). Amino acid sequences of seven proteolytic peptide fragments of MNBP-B were determined, and the cDNA clones were isolated. MNBP-B was proven identical to the single-stranded DNA-binding protein, UP1. Recombinant UP1 bound to single-stranded d(GGCAG)(n) and other G-rich repetitive sequences, such as d(GTCAGG)(n) and d(GTTAGG)(n). In addition, UP1 was demonstrated by CD spectrum analysis to unfold the intramolecular quadruplex structure of d(GGCAG)(5) and d(TTAGGG)(4) and to abrogate the arrest of DNA synthesis at the d(GGG)(n) site. This ability of UP1 suggests that unfolding of quadruplex DNA is required for DNA synthesis processes.