NMR structure of the hRap1 Myb motif reveals a canonical three-helix bundle lacking the positive surface charge typical of Myb DNA-binding domains.

Mammalian telomeres are composed of long tandem arrays of double-stranded telomeric TTAGGG repeats associated with the telomeric DNA-binding proteins, TRF1 and TRF2. TRF1 and TRF2 contain a similar C-terminal Myb domain that mediates sequence-specific binding to telomeric DNA. In the budding yeast, telomeric DNA is associated with scRap1p, which has a central DNA-binding domain that contains two structurally related Myb domains connected by a long linker, an N-terminal BRCT domain, and a C-terminal RCT domain. Recently, the human ortholog of scRap1p (hRap1) was identified and shown to contain a BRCT domain and an RCT domain similar to scRap1p. However, hRap1 contained only one recognizable Myb motif in the center of the protein. Furthermore, while scRap1p binds telomeric DNA directly, hRap1 has no DNA-binding ability. Instead, hRap1 is tethered to telomeres by TRF2. Here, we have determined the solution structure of the Myb domain of hRap1 by NMR. It contains three helices maintained by a hydrophobic core. The architecture of the hRap1 Myb domain is very close to that of each of the Myb domains from TRF1, scRap1p and c-Myb. However, the electrostatic potential surface of the hRap1 Myb domain is distinguished from that of the other Myb domains. Each of the minimal DNA-binding domains, containing one Myb domain in TRF1 and two Myb domains in scRap1p and c-Myb, exhibits a positively charged broad surface that contacts closely the negatively charged backbone of DNA. By contrast, the hRap1 Myb domain shows no distinct positive surface, explaining its lack of DNA-binding activity. The hRap1 Myb domain may be a member of a second class of Myb motifs that lacks DNA-binding activity but may interact instead with other proteins. Other possible members of this class are the c-Myb R1 Myb domain and the Myb domains of ADA2 and Adf1. Thus, while the folds of all Myb domains resemble each other closely, the function of each Myb domain depends on the amino acid residues that are located on the surface of each protein.     

Solution structure of a telomeric DNA complex of human TRF1.

BACKGROUND: Mammalian telomeres consist of long tandem arrays of double-stranded TTAGGG sequence motif packaged by TRF1 and TRF2. In contrast to the DNA binding domain of c-Myb, which consists of three imperfect tandem repeats, DNA binding domains of both TRF1 and TRF2 contain only a single Myb repeat. In a DNA complex of c-Myb, both the second and third repeats are closely packed in the major groove of DNA and recognize a specific base sequence cooperatively. RESULTS: The structure of the DNA binding domain of human TRF1 bound to telomeric DNA has been determined by NMR. It consists of three helices, whose architecture is very close to that of three repeats of the c-Myb DNA binding domain. Only the single Myb domain of TRF1 is sufficient for the sequence-specific recognition. The third helix of TRF1 recognizes the TAGGG part in the major groove, and the N-terminal arm interacts with the TT part in the minor groove. CONCLUSIONS: The DNA binding domain of TRF1 can specifically and fully recognize the AGGGTT sequence. It is likely that, in the dimer of TRF1, two DNA binding domains can bind independently in tandem arrays to two binding sites of telomeric DNA that is composed of the repeated AGGGTT motif. Although TRF2 plays an important role in the t loop formation that protects the ends of telomeres, it is likely that the binding mode of TRF2 to double-stranded telomeric DNA is almost identical to that of TRF1.     

Interaction of a protooncogene product, Myb with DNAs.

The DNA-binding domain of Myb consists of three imperfect tandem repeats and the third one which is essential for sequence-specific binding was established to have a helix-turn-helix-related motif. DNA sequences recognized by Myb have been reported to contain TAACPy sequence. Here we have examined the details of Myb-binding sequence. Using DNAs with a single mutation on the various sites of two specific DNAs and some fragments of the DNA-binding domain of Myb, we have found that (i) in a specific DNA which contains only one AAC sequence, each AAC nucleotide is found to be essential for the specific binding of Myb, while any other mutations cause no serious binding loss, (ii) in a specific DNA which contains two AAC sequences separately, one AAC is not so important in the binding, and (iii) for the specific binding with DNA, at least both repeats 2 and 3 of Myb are required. These findings suggest that repeat 3 containing a helix-turn-helix-related structure recognizes the core AAC sequence and repeat 2 supports this recognition by interactions with phosphate groups of DNA.     

Comparison between TRF2 and TRF1 of their telomeric DNA-bound structures and DNA-binding activities

Mammalian telomeres consist of long tandem arrays of double-stranded telomeric TTAGGG repeats packaged by the telomeric DNA-binding proteins TRF1 and TRF2. Both contain a similar C-terminal Myb domain that mediates sequence-specific binding to telomeric DNA. In a DNA complex of TRF1, only the single Myb-like domain consisting of three helices can bind specifically to double-stranded telomeric DNA. TRF2 also binds to double-stranded telomeric DNA. Although the DNA binding mode of TRF2 is likely identical to that of TRF1, TRF2 plays an important role in the t-loop formation that protects the ends of telomeres. Here, to clarify the details of the double-stranded telomeric DNA-binding modes of TRF1 and TRF2, we determined the solution structure of the DNA-binding domain of human TRF2 bound to telomeric DNA; it consists of three helices, and like TRF1, the third helix recognizes TAGGG sequence in the major groove of DNA with the N-terminal arm locating in the minor groove. However, small but significant differences are observed; in contrast to the minor groove recognition of TRF1, in which an arginine residue recognizes the TT sequence, a lysine residue of TRF2 interacts with the TT part. We examined the telomeric DNA-binding activities of both DNA-binding domains of TRF1 and TRF2 and found that TRF1 binds more strongly than TRF2. Based on the structural differences of both domains, we created several mutants of the DNA-binding domain of TRF2 with stronger binding activities compared to the wild-type TRF2.     

Potential role for 53BP1 in DNA end-joining repair through direct interaction with DNA

Upon DNA damage, p53-binding protein 1 (53BP1) relocalizes to sites of DNA double-strand breaks and forms discrete nuclear foci, suggesting its role in DNA damage responses. We show that 53BP1 changed its localization from the detergent soluble to insoluble fraction after treatment of cells with x-ray, but not with ultraviolet or hydroxyurea. Either DNase or phosphatase treatment of the insoluble fraction released 53BP1 into the soluble fraction, showing that 53BP1 binds to chromatin in a phosphorylation-dependent manner after X-irradiation of cells. 53BP1 was retained at discrete nuclear foci in X-irradiated cells even after detergent extraction of cells, showing that the chromatin binding of 53BP1 occurs at sites of DNA double-strand breaks. The minimal domain for focus formation was identified by immunofluorescence staining of cells ectopically expressed with 53BP1 deletion mutants. This domain consisted of conserved Tudor and Myb motifs. The Tudor plus Myb domain possessed chromatin binding activity in vivo and bound directly to both double-stranded and single-stranded DNA in vitro. This domain also stimulated end-joining by DNA ligase IV/Xrcc4, but not by T4 DNA ligase in vitro. We conclude that 53BP1 has the potential to participate directly in the repair of DNA double-strand breaks.     

DNA and redox state induced conformational changes in the DNA-binding domain of the Myb oncoprotein.

The DNA-binding domain of the oncoprotein Myb comprises three imperfect repeats, R1, R2 and R3. Only R2 and R3 are required for sequence-specific DNA-binding. Both are assumed to contain helix-turn-helix (HTH)-related motifs, but multidimensional heteronuclear NMR spectroscopy revealed a disordered structure in R2 where the second HTH helix was predicted [Jamin et al. (1993) Eur. J. Biochem., 216, 147-154]. We propose that the disordered region folds into a 'recognition' helix and generates a full HTH-related motif upon binding to DNA. This would move Cys43 into the hydrophobic core of R2. We observed that Cys43 was accessible to N-ethylmaleimide alkylation in the free protein, but inaccessible in the DNA complex. Mutant proteins with charged (C43D) or polar (C43S) side chains in position 43 bound DNA with reduced affinity, while hydrophobic replacements (C43A, C43V and C43I) gave unaltered or improved DNA-binding. Specific DNA-binding enhanced protease resistance dramatically. Fluorescence emission spectra and quenching experiments supported a DNA-induced conformational change. Moreover, reversible oxidation of Cys43 had an effect similar to the inactivating C43D mutation. The highly oxidizable Cys43 could function as a molecular sensor for a redox regulatory mechanism turning specific DNA-binding on or off by controlling the DNA-induced conformational change in R2.     

Solution structure of telomere binding domain of AtTRB2 derived from Arabidopsis thaliana

Telomere homeostasis is regulated by telomere-associated proteins, and the Myb domain is well conserved for telomere binding. AtTRB2 is a member of the SMH (Single-Myb-Histone)-like family in Arabidopsis thaliana, having an N-terminal Myb domain, which is responsible for DNA binding. The Myb domain of AtTRB2 contains three α-helices and loops for DNA binding, which is unusual given that other plant telomere-binding proteins have an additional fourth helix that is essential for DNA binding. To understand the structural role for telomeric DNA binding of AtTRB2, we determined the solution structure of the Myb domain of AtTRB2 (AtTRB2_1–64) using nuclear magnetic resonance (NMR) spectroscopy. In addition, the inter-molecular interaction between AtTRB2_1–64 and telomeric DNA has been characterized by the electrophoretic mobility shift assay (EMSA) and NMR titration analyses for both plant (TTTAGGG)n and human (TTAGGG)n telomere sequences. Data revealed that Trp28, Arg29, and Val47 residues located in Helix 2 and Helix 3 are crucial for DNA binding, which are well conserved among other plant telomere binding proteins. We concluded that although AtTRB2 is devoid of the additional fourth helix in the Myb-extension domain, it is able to bind to plant telomeric repeat sequences as well as human telomeric repeat sequences.     

Roles of active site residues and the HUH motif of the F plasmid TraI relaxase

Bacterial conjugation, transfer of a single strand of a conjugative plasmid between bacteria, requires sequence-specific single-stranded DNA endonucleases called relaxases or nickases. Relaxases contain an HUH (His-hydrophobe-His) motif, part of a three-His cluster that binds a divalent cation required for the cleavage reaction. Crystal structures of the F plasmid TraI relaxase domain, with and without bound single-stranded DNA, revealed an extensive network of interactions involving HUH and other residues. Here we study the roles of these residues in TraI function. Whereas substitutions for the three His residues alter metal-binding properties of the protein, the same substitution at each position elicits different effects, indicating that the residues contribute asymmetrically to metal binding. Substitutions for a conserved Asp that interacts with one HUH His demonstrate that the Asp modulates metal affinity despite its distance from the metal. The bound metal enhances binding of ssDNA to the protein, consistent with a role for the metal in positioning the scissile phosphate for cleavage. Most substitutions tested caused significantly reduced in vitro cleavage activities and in vivo transfer efficiencies. In summary, the results suggest that the metal-binding His cluster in TraI is a finely tuned structure that achieves a sufficient affinity for metal while avoiding the unfavorable electrostatics that would result from placing an acidic residue near the scissile phosphate of the bound ssDNA.     

Independence of metal binding between tandem Cys2His2 zinc finger domains.

Most Cys2His2 zinc finger proteins contain tandem arrays of metal binding domains. The tandem nature of these arrays suggests that metal binding by these domains may not be independent but rather that metal binding may occur in a cooperative manner. This is especially true in light of the crystal structure of a three zinc finger array bound to DNA that revealed several types of interactions between domains. To address this question, peptides containing two tandem domains have been prepared. While metal binding studies do show that the two finger peptide has a metal ion affinity about threefold higher than that for a single domain peptide with the same sequence, additional studies reveal that this behavior is due to increased single site affinities in the context of the two domain peptide rather than to cooperativity. These studies indicate that domains of this type are independent of one another with regard to metal binding, at least in the absence of DNA. This observation has implications with regard to the question of whether the activities of proteins of this class might be modulated by available zinc concentrations.