Yeast Nhp6A/B and Mammalian Hmgb1 Facilitate the Maintenance of Genome Stability

AbstractSaccharomyces cerevisiae Nhp6A and Nhp6B are chromatin architectural factors that belong to the high-mobility group box (HMGB) superfamily and appear to be functionally related to mammalian Hmgb1 [1]. They bind to the minor groove of double-stranded DNA in a non-sequence-specific manner [2] and thereby influence chromatin structure [3]. Previous work has implicated these proteins in a variety of nuclear processes, including chromatin remodeling, DNA replication, transcription, and recombination [4–10]. Here, we show that Nhp6A/B loss leads to increased genomic instability, hypersensitivity to DNA-damaging agents, and shortened yeast cell life span that is associated with elevated levels of extrachromosomal rDNA circles. Furthermore, we show that hypersensitivity toward UV light does not appear to reflect a decreased capacity for DNA repair but instead correlates with higher levels of UV-induced thymine dimer adducts being formed in cells lacking Nhp6A/B. Likewise, we show that mouse fibroblasts lacking Hmgb1 display higher rates of damage after UV irradiation than wild-type controls and also exhibit pronounced chromosomal instability. Taken together, these data indicate that Nhp6A/B and Hmgb1 protect DNA from damaging agents and thus guard against the generation of genomic aberrations

Helical properties of micelle-bound Hsp12.

<p>(A) The four α-helices are represented as ribbons and colour coded from the N-terminus (blue) to the C-terminus (red) in a representative structure. (B,C) Analysis of charge distribution with hydrophobic residues labelled green and charged residues labelled red in both ribbon (B) and surface (C) representation, illustrating the amphipathic nature of Hsp12. Structures were generated using Chimera.</p

Backbone dynamics and chemical shift-based secondary structure of Hsp12.

<p><i>T</i>₁, <i>T</i>₂ and <i>T</i>₁/<i>T</i>₂ relaxation values are shown for Hsp12 in the presence (A,C,E) and absence (B,D,F) of 100 mM SDS at 318 K. <i>T</i>₁ and <i>T</i>₂ relaxation times for micelle-bound (A,C) Hsp12 show significant variation; contrasting with the similar relaxation values observed for free Hsp12 (B,D). Micelle-bound Hsp12 (E) shows grouped variations in the <i>T</i>₁/<i>T</i>₂ values ranging from approximately 1.5 to 14, indicating a wide range of mobility and a clear differentiation of secondary structure elements; whereas the free form (F) shows consistent values of around 2, indicating a completely unstructured protein. (G) The assigned chemical shifts at 318 K in 100 mM SDS expressed as deviation from random coil are shown aligned with the primary sequence and the positions of the α-helices.</p

Ensemble of structures calculated for micelle-bound Hsp12 overlaid on each of the four helices.

<p>Ensemble of twenty structures overlaid on helices I (A), II (B), III (C) and IV (D). No long-range interactions were detected and so the helices appear free to move independently with no overall fold being evident.</p

DR induces expression of a relatively small number of proteins.

<p>Wild type BY4741 yeast cells were grown in standard (2% glucose) and DR (0.5% glucose) conditions before lysis and separation of proteins by 2-D electrophoresis. Wide-range (pH 3–10) gels revealed no obvious reproducible differences in protein expression, as illustrated by representative gels shown in panel (A). Narrow pH range gels (pH 3–5.6 and 5.3–6.5) revealed changes in protein spots, which were identified by mass spectrometry. Selected identified proteins are indicated by arrows in panels (B) and (C).</p

Hsp12 is unstructured in solution, but folds in the presence of SDS.

<p>(A) ¹H-¹⁵N HSQC spectrum of Hsp12 in aqueous solution at 298 K. The spectrum shows only sharp peaks with random coil shifts indicating the absence of any structured regions. (B) ¹H-¹⁵N HSQC spectrum of Hsp12 at 303 K in the presence of increasing concentrations of SDS (0, 1, 2, 5, 8 mM Red -> Blue). SDS causes a considerable increase in the amount of chemical shift dispersion implying increased levels of folded material/regions. (C) Assigned ¹H-¹⁵N HSQC spectrum of Hsp12 at 318 K in the presence of 100 mM SDS.</p