A mammalian high mobility group protein recognizes any stretch of six A.T base pairs in duplex DNA.

Abstract

ABSTRACTa-Protein is a high mobility group protein originally purified from African green monkey cells based on its affinity for the 172-base-pair repeat of monkey a-satellite DNA. We have used DNase I footprinting to identify 50 a-protein binding sites on simian virus 40 DNA and thereby to determine the DNA binding specificity of this mammalian nuclear protein. a-Protein binds with approximately equal affinity to any run of six or more APT base pairs in duplex DNA, to many, if not all, runs of five AkT base pairs, and to a small number of other sequences within otherwise (A+T)-rich regions. Unlike well characterized sequence-specific DNA binding proteins such as bacterial repressors, a-protein makes extensive contacts within the minor groove of B-DNA. These and related findings indicate that, rather than binding to a few specific DNA sequences, a-protein recognizes a configuration of the minor groove characteristic of short runs of AT base pairs. We discuss possible functions of a-protein and the similarities in DNA recognition by a-protein and the antibiotic netropsin.Previous studies from this laboratory have addressed the existence and properties of DNA sequence-specific nucleosome-binding proteins (1-3). In particular, we searched for a protein specific for the a-satellite DNA (a-DNA) of the African green monkey. Using the "band-competition" assay, a generally applicable electrophoretic assay for specific DNA-binding proteins in crude extracts, we purified an abundant nuclear protein from green monkey CV-1 cells that preferentially bound to a-DNA (1). The solubility properties, amino acid composition, and primary structure of this =10 kDa protein (tentatively called a-protein) operationally classified it as a high mobility group (HMG) protein (4-6), distinct from the other major HMG proteins, HMG 1, -2, -14, and -17 (J. McCartney, F.S., M.J.S., J. Smart, and A.V., unpublished data). The preferred a-nucleosome frame detected in isolated chromatin (7,8) is precisely bordered by a-protein binding sites (GATAT'IT) on a-DNA, suggesting that aprotein might function as a nucleosome-positioning or phasing protein (1).To address the binding specificity of a-protein in more detail, we mapped a-protein binding sites on simian virus 40 (SV40) DNA. a-Protein binds with approximately equal affinity not only to the GATATTT sequences in SV40 DNA but also to >50 other sites in the -2.4 kilobase pairs (kbp) that we have examined by DNase I footprinting. Thus, rather than recognizing a few specific nucleotide sequences, aprotein recognizes an aspect of B-DNA conformations, most likely a configuration of the minor groove, that is characteristic of short runs of APT base pairs.These and other properties of a-protein set it apart from the more extensively 'studied prokaryotic and eukaryotic sequence-specific DNA binding proteins, whose characteristic features include the predominance of major groove interactions and little or no sequence degeneracy in DNA recognition (9). MATERIALS AND METHODSDNase I Footprinting. DNA fragments ...

The nonspecific DNA-binding and -bending proteins HMG1 and HMG2 promote the assembly of complex nucleoprotein structures.

“…By measuring the efficiency of invertasome assembly, we also did not find that HU or HMG1 was altering the helical repeat of supercoiled DNA (Haykinson and Johnson 1993; data not shown). A significant preference of HMG1 or HMG2 activity on AT-rich regions was also not observed by either Hin-mediated inversion or ligation assays, in contrast to a report on chicken HMG2a (Brown and Anderson 1986) and another subgroup of HMG proteins, HMGI/Y, which bind preferentially to runs of five or more A or T nucleotides (Solomon et al 1986;Russnak et al 1988;Thanos and Maniatis 1992).…”

“…Because A-tracts are associated with intrinsically curved D N A (for review, see Travers and Klug 1990;Hagerman 1992), and some members of the HMG protein family--HMGI/Y and chicken H M G 2 a --are known to preferentially bind to AT-rich regions of DNA (Brown and Anderson 1986;Solomon et al 1986;Russnak et al 1988;Thanos and Maniatis 1992), we wanted to determine whether the A-tracts in our recombination substrates influence the function of HMG proteins in these assays. For this purpose we used a substrate whose intervening segment is 63% GC-rich and contains only two AA or TT dinucleotides (pMS551-97.1).…”

A complex DNA-repeat structure within the Selenoprotein P promoter contains a functionally relevant polymorphism and is genetically unstable under conditions of mismatch repair deficiency

“…In cells with defective mismatch repair, SSRs are prone to develop genetic instability, so called microsatellite instability, as a result of insufficient DNA mismatch repair of spontaneous mutations in repeat motifs due to strand mispairing during DNA replication. 28 SSRs like poly(T) repeats 29 and poly(TC) repeats 30 can function as binding sites for regulatory proteins in upstream activation sequences. In several studies, deletion of SSRs from upstream promoter regions has been shown to reduce or eliminate transcriptional activity, whereas expansion of SSRs led to an increase of transcriptional activity, possibly due to alterations of binding of regulatory proteins.…”

A retrospective study of high mobility group protein I(Y) as progression marker for prostate cancer determined by in situ hybridization

“…We and others have shown that loss of E-cadherin, a molecule involved in maintaining tissue integrity, is associated with progression in prostate cancer (Umbas et al, 1992(Umbas et al, , 1994 al., 1988, 1989) and the closely related HMGI-C (Manfioletti et al, 1991). Members of the HMG-I(Y) family are distinguished from other groups of HMG proteins by their ability to specifically bind to the minor groove of A: T-rich DNA sequences (Elton et al, 1986;Solomon et al, 1986), presumably similar to antitumour and antiviral drugs (netropsin, distamicin) and the dye Hoechst 33258 (Disney et al, 1989;Wegner et al, 1990). …”