Boundaries and physical characterization of a new domain shared between mammalian 53BP1 and yeast Rad9 checkpoint proteins

Abstract

Eukaryotic cells have evolved DNA damage checkpoints in response to genome damage. They delay the cell cycle and activate repair mechanisms. The kinases at the heart of these pathways and the accessory proteins, which localize to DNA lesions and regulate kinase activation, are conserved from yeast to mammals. For Saccharomyces cerevisiae Rad9, a key adaptor protein in DNA damage checkpoint pathways, no clear human ortholog has yet been described in mammals. Rad9, however, shares localized homology with both human BRCA1 and 53BP1 since they all contain tandem C-terminal BRCT (BRCA1 C-terminal) motifs. 53BP1 is also a key mediator in DNA damage signaling required for cell cycle arrest, which has just been reported to possess a tandem Tudor repeat upstream of the BRCT motifs. Here we show that the major globular domain upstream of yeast Rad9 BRCT domains is structurally extremely similar to the Tudor domains recently resolved for 53BP1 and SMN. By expressing several fragments encompassing the Tudor-related motif and characterizing them using various physical methods, we isolated the independently folded unit for yeast Rad9. As in 53BP1, the domain corresponds to the SMN Tudor motif plus the contiguous HCA predicted structure region at the C terminus. These domains may help to further elucidate the structural and functional features of these two proteins and improve knowledge of the proteins involved in DNA damage.

Figure 1.

(A). Schematic representations of S. cerevisiae Rad9, human 53BP1, and human BRCA1 primary sequence showing the location of conserved domains: (i) for ScRad9, two C-terminal BRCT domains—an [S/T]Q cluster domain (SCD) and a Chk1 activation domain (CAD). The region reported to interact with Rad53 FHA2 is indicated by a black horizontal line. (ii) for h53BP1, the PSI-Blast-predicted Tudor domain and two C-terminal BRCT domains. The regions reported to allow 53BP1 to form foci in response to DNA damage, and to bind to phosphorylated H2AX (-H2AX) in vitro, are indicated by a black horizontal line. (iii) for hBRCA1, two C-terminal BRCT domains, an SQ cluster domain, and an N-terminal RING finger domain. (B) Hydrophobic cluster analysis plots of the regions ScRad9[724–977] and m53BP1[1433–1637], with the positions of the experimental constructs. m53BP1[1433–1637] is 94% identical to the corresponding h53BP1 [1448–1652] segment. The standard one-letter code for amino acids is used except for proline (regular secondary structure breaker), glycine (the less constrained amino acid), serine, and threonine, which are represented by a star, a diamond, an empty rectangle, and a rectangle containing a black rectangle, respectively.

Figure 2.

Multiple alignment and hydrophobic cluster analysis plots of the Tudor and Tudor putative motif of hSMN, m53BP1, and ScRad9. (A) ClustlalW multialignment of hSMN[90–149] Tudor domain, m53BP1[1466–1535] PSI-Blast-predicted Tudor motif, and ScRad9[764–855] putative Tudor motif. Secondary structure predictions are indicated below each sequence. e, extended or β-strand structure; c, coiled structure; h, helix. (B) HCA plots of hSMN[90–149], m53BP1[1466–1535], and ScRad9[764–855] . The vertical lines are based on the multialignment and correspond to four conserved parts: a, b, c, and d. The orange circles represent amino acids displaying similarity, and the red circles show conserved amino acids.

Figure 3.

Physicochemical characteristics of Tudor-related fragments. (A) Size-exclusion chromatography of Tudor-related fragments from ScRad9 and m53BP1. The elution volume vs. molecular mass is on a logarithmic scale. The column (Superdex 75) was calibrated with globular proteins (+): ribonuclease A (13.7 kDa), chymotrypsin (25 kDa), ovalbumin (43 kDa), and albumin (60 kDa). The standard curve was drawn from molecular weight marker data, whereas the experimental elution volume of each fragment is spotted on the abscissa scale.Δ, m53BP1[1463–1617]; □, Stag-m53BP1[1466–1535]; ▴, ScRad9 [754–947]; •, Stag-ScRad9[754–947]; ▪, ScRad9[764–947]; ♦, Stag-ScRad9[764–855]. (B) Far-UV CD spectra of (1) m53BP1[1463–1617], (2) ScRad9[754–947], (3) Stag ScRad9[754–947], (4) Stag ScRad9[764–855], and (5) Stag-m53BP1[1466–1535]. (C) Microcalorimetric heat transitions are depicted as dependence of heat capacity on temperature for (1) m53BP1[1463–1617], (2) ScRad9[754– 947], (3) Stag-ScRad9[754–947], and (4) Stag-ScRad9[764–855]. Concentration of the heated protein solutions was 0.05mg•mL⁻¹ (i.e., 2.9 μM, 2.2 μM, 1.95 μM, and 3.6 μM, respectively). Heat transitions exist for 1, 2, and 3.

Figure 4.

¹H-¹⁵N Heteronuclear Single-Quantum Coherence (HSQC) spectra. (A) Spectrum of m53BP1[1463–1617]. (B) Spectrum of ScRad9[754– 947].