The Saccharomyces cerevisiae High Mobility Group Box Protein HMO1 Contains Two Functional DNA Binding Domains

Abstract

High mobility group box (HMGB) proteins are architectural proteins whose HMG DNA binding domains confer significant preference for distorted DNA, such as 4-way junctions. HMO1 is one of 10 Saccharomyces cerevisiae HMGB proteins, and it is required for normal growth and plasmid maintenance and for regulating the susceptibility of yeast chromatin to nuclease. Using electrophoretic mobility shift assays, we have shown here that HMO1 binds 26-bp duplex DNA with K d ‫؍‬ 39.6 ؎ 5.0 nM and that its divergent box A domain participates in DNA interactions, albeit with low affinity. HMO1 has only modest preference for DNA with altered conformations, including DNA with nicks, gaps, overhangs, or loops, as well as for 4-way junction structures and supercoiled DNA. HMO1 binds 4-way junctions with half-maximal saturation of 19.6 ؎ 2.2 nM, with only a modest increase in affinity in the absence of magnesium ions (half-maximal saturation 6.1 ؎ 1.1 nM). Whereas the box A domain contributes modest structure-specific binding, the box B domain is required for high affinity binding. HMO1 bends DNA, as measured by DNA cyclization assays, facilitating cyclization of 136-, 105-, and 87-bp DNA, but not 75-bp DNA, and it has a significantly longer residence time on DNA minicircles compared with linear duplex DNA. The unique DNA binding properties of HMO1 are consistent with global roles in the maintenance of chromatin structure. High mobility group (HMG)1 proteins constitute a significant proportion of non-histone proteins of eukaryotic chromatin. They are abundant proteins that are grouped, in part based on their DNA binding characteristics, into three major classes, HMGA, HMGB, and HMGN (1-4). HMGB proteins contain one or more homologous repeats of the ϳ80-amino acid sequence HMG box and are classified into two families based on the abundance, function, and DNA specificity of this conserved region (1, 5, 6). The moderately sequence-specific family is typified by transcription factors such as sex-determining factor SRY and lymphoid enhancer factor LEF-1 (7, 8), whereas the non-sequence-specific family is represented by so-called architectural factors HMGB1/2 and the Saccharomyces cerevisiae non-histone chromosomal proteins 6A and 6B (NHP6A/B) (9).The tertiary structures of HMG boxes from sequence-specific and non-sequence-specific proteins have revealed an evolutionarily conserved, common global fold consisting of an L-shaped structure composed of three ␣-helices (10 -19). The HMG DNA binding domain, which interacts with ϳ10 bp of duplex, binds to the minor groove of DNA by partial intercalation of one or two surface-exposed, conserved hydrophobic residues into the base pair stack. Consequently, the DNA is greatly distorted, resulting in a sharp bend and helical underwinding (4,8,14,16,20). SRY and LEF-1 cause bending by insertion of helix I hydrophobic residues Ile and Met, respectively, into the base pair stack (Fig. 1) (8, 17). HMGB1 contains tandem HMG box domains referred to as box A and box B; DNA-intercalating residues are located...

Yeast HMO1: Linker Histone Reinvented

“…1). Box A, which has only limited similarity to consensus HMG domains, functions as a dimerization domain; it has a low affinity for DNA but exhibits some structural specificity, including preferred binding to four-way junction DNA, whereas canonical box B has a higher affinity for DNA but a lower structural specificity (130,132). The HMO1 box A domain contributes to DNA bending; in contrast, the box B domain contributes most of the DNA binding affinity but fails to bend linear DNA (133).…”

Both HMG boxes in Hmo1 are essential for DNA binding in vitro and in vivo

“…21,22) In contrast to these findings, a mutation in box A of Hmo1 (T30R) identified in the genetic screen abolished the DNA binding activity of Hmo1 both in vivo and in vitro (Fig. 3(B) and (C)).…”

“…For example, while box A has only a slight contribution to the DNA binding activity of Hmo1 compared with that of box B, it is responsible for structure-specific DNA binding of the protein, albeit less significantly. 21) Furthermore, box A may interact with the carboxy-terminal tail of Hmo1 to bend DNA, 22) or with box A itself to form dimer or oligomer. 32) However, the significance of these properties of box A in vivo has not been verified.…”

“…The fact that mutations affecting the DNA binding ability of Hmo1 were located at or close to the first α-helix of box B is consistent with the previously held notion that box B is responsible for most of the DNA binding activity of Hmo1. 21,22) On the other hand, two large α-helices were also predicted within box A of Hmo1 (Supplemental Fig. 5, amino acids 7-41 (1st) and 45-63 (2nd)).…”

“…Previous in vitro studies suggested that box B is responsible for most of the DNA binding activity of Hmo1, whereas the contribution of box A to this activity is very limited. 21,22) It was reported recently that the integrity of box A is required for the function of Hmo1 in the RNA polymerase I transcription in vivo, whereas the requirement of box A in the transcription of RPG (such as RPS23A) by RNA polymerase II is unclear. 23) In contrast to these previous studies, the box A mutations identified in the genetic screen performed here impaired the ability of Hmo1 to bind to DNA.…”