γH2A binds Brc1 to maintain genome integrity during S‐phase

ATM^Tel1 and ATR^Rad3 checkpoint kinases phosphorylate the C‐terminus of histone H2AX (H2A in yeasts) in chromatin flanking DNA damage, establishing a recruitment platform for checkpoint and repair proteins. Phospho‐H2A/X (γH2A/X)‐binding proteins at double‐strand breaks (DSBs) have been characterized, but those required for replication stress responses are unknown. Here, we present genetic, biochemical, small angle X‐ray scattering (SAXS), and X‐ray structural studies of the Schizosaccharomyces pombe Brc1, a 6‐BRCT‐domain protein that is structurally related to Saccharomyces cerevisiae Rtt107 and mammalian PTIP. Brc1 binds γH2A to form spontaneous and DNA damage‐induced nuclear foci. Spontaneous Brc1 foci colocalize with ribosomal DNA repeats, a region prone to fork pausing and genomic instability, whereas DNA damage‐induced Brc1 foci colocalize with DSB response factors. γH2A binding is critical for Brc1 function. The 1.45 Å resolution crystal structure of Brc1–γH2A complex shows how variable BRCT insertion loops sculpt tandem‐BRCT phosphoprotein‐binding pockets to facilitate unique phosphoprotein‐interaction specificities, and unveils an acidic DNA‐mimicking Brc1 surface. From these results, Brc1 docking to γH2A emerges as a critical chromatin‐specific response to replication‐associated DNA damage.     

Histone h1(0) and its carboxyl-terminal domain bind in the major groove of DNA

The binding of histone H1(0) to T4 bacteriophage DNA was investigated using thermal denaturation of the DNA titrated with varying concentrations of protein. The H1(0) used was expressed in and purified from a strain of E. coli and is therefore homogeneous with respect to H1 subtype and posttranslational modifications. Two types of T4 DNA were used: wild-type, which contains a modification of the cytosine residues that projects into the major groove: and a mutant type, which lacks the modification of the cytosines. Data were compared to simulated thermal denaturation curves to determine estimates for binding affinity and binding site size in base pairs of the protein. Analysis of the data yielded values of 10(8) M(-1) for K, the binding affinity, and 10 base pairs for n, the number of base pairs covered by one protein, for the mutant T4 DNA. Analysis of the wild-type DNA data suggested that the glucose projecting into the major groove of this DNA decreases the number of sites to which the H1(0) protein can bind, indicating that there are interactions between the protein and the major groove of DNA. The binding site size on this DNA is 10 base pairs, the same as on the unmodified DNA. The affinity for wild-type DNA is slightly higher, 10(9) M(-1). Data were collected and analyzed for binding of two domains of the protein as well, the carboxyl-terminal domain and the central globular domain. Binding of the carboxyl-terminal domain was quantitatively and qualitatively similar to that of the full-length protein. In contrast, binding of the globular domain was quite different: it binds much more weakly, with a K of 6 x 10(4) M(-1), and covers fewer base pairs, with an n of 3. Also, there was no evidence that the globular domain interacts with the major groove of DNA.     

Calcium binding to the isolated beta-hydroxyaspartic acid-containing epidermal growth factor-like domain of bovine factor X

Coagulation factor X is a vitamin K-dependent protein composed of discrete domains or modules. A proteolytically modified derivative of factor X that lacks the NH2-terminal gamma-carboxyglutamic acid (Gla)-containing region retains one Ca2+ binding site. To localize this Gla-independent Ca2+ binding site and to facilitate future studies aimed at elucidating structure-function relationship in the factor X molecule, we have devised a method to isolate the first beta-hydroxyaspartic acid (Hya)-containing epidermal growth factor (EGF)-like domain from proteolytic digests of bovine factor X performed under strictly controlled conditions. The EGF-like domain, corresponding to residues 45-86 in bovine factor X, was obtained in more than 50% recovery, and was at least 98% homogeneous as judged by NH2-terminal sequence analysis. Ca2+ binding to the isolated EGF-like domain was studied by 1H NMR spectroscopy. On binding of Ca2+ to the domain the resonances from Tyr-68 centered at 6.8 ppm were affected. The Ca2+ concentration dependence of the chemical shift was used to calculate the Ca2+ binding constant, resulting in a K alpha of 4 X 10(3) M-1 at pH 8.5 and 1 X 10(3) M-1 at pH 7.4, the higher value presumably reflecting an increase in negative surface charge due to deprotonation of a histidine residue with a pK alpha of 7.4. The NMR spectra gave no evidence of a conformational change in the EGF-like domain between pH 6 and 8.5.     

Mutational analysis of arginine 276 in the leucine-loop of human uracil-DNA glycosylase

Uracil residues are eliminated from cellular DNA by uracil-DNA glycosylase, which cleaves the N-glycosylic bond between the uracil base and deoxyribose to initiate the uracil-DNA base excision repair pathway. Co-crystal structures of the core catalytic domain of human uracil-DNA glycosylase in complex with uracil-containing DNA suggested that arginine 276 in the highly conserved leucine intercalation loop may be important to enzyme interactions with DNA. To investigate further the role of Arg(276) in enzyme-DNA interactions, PCR-based codon-specific random mutagenesis, and site-specific mutagenesis were performed to construct a library of 18 amino acid changes at Arg(276). All of the R276X mutant proteins formed a stable complex with the uracil-DNA glycosylase inhibitor protein in vitro, indicating that the active site structure of the mutant enzymes was not perturbed. The catalytic activity of the R276X preparations was reduced; the least active mutant, R276E, exhibited 0.6% of wildtype activity, whereas the most active mutant, R276H, exhibited 43%. Equilibrium binding studies utilizing a 2-aminopurine deoxypseudouridine DNA substrate showed that all R276X mutants displayed greatly reduced base flipping/DNA binding. However, the efficiency of UV-catalyzed cross-linking of the R276X mutants to single-stranded DNA was much less compromised. Using a concatemeric [(32)P]U.A DNA polynucleotide substrate to assess enzyme processivity, human uracil-DNA glycosylase was shown to use a processive search mechanism to locate successive uracil residues, and Arg(276) mutations did not alter this attribute.     

Identity of the RNA-binding protein K of hnRNP particles with protein H16, a sequence-specific single strand DNA-binding protein.

Protein H16, which we have identified previously in mammalian cell lines, binds in vitro to two single stranded DNA sites on the late strand of the early promoter of SV40. It has no other single strand binding site in the SV40 genome and does not bind to double stranded DNA. In vitro, H16 can be shown to stimulate strongly the activity of purified RNA polymerase II. Here we have purified this 70 kDa protein from cultured monkey cells and have sequenced three of its tryptic peptides. The analysis indicates that H16 is the simian homolog of human protein K, a nuclear RNA-binding protein found in heterogeneous nuclear ribonucleoprotein (hnRNP) particles, which contains a KH domain present in several proteins including the fragile X mental retardation gene product (FMR1). The binding affinities of protein K/H16 for RNA and DNA were subsequently compared in detail. They showed that under conditions where K/H16 binds strongly to its single stranded DNA site, it binds very weakly to the corresponding RNA sequence. This result suggests a possible shuttling of the protein from RNA to DNA during processes which involve opening of the DNA double helix.     

Mapping of functional and antigenic domains of the alpha 4 protein of herpes simplex virus 1.

Monoclonal antibodies to alpha 4, the major regulatory protein of herpes simplex virus 1, have been shown to differ in their effects on the binding of the protein to its DNA-binding site in the promoter-regulatory domain of an alpha gene. To map the epitopes, we expressed truncated genes in transient expression systems. All 10 monoclonal antibodies tested reacted with the N-terminal 288-amino-acid polypeptide. To map the epitopes more precisely, 29 15-mer oligopeptides, overlapping by five amino acids at each end, were synthesized and reacted with the monoclonal antibodies. The nine reactive monoclonal antibodies were mapped to seven sites. Of the two monoclonal antibodies which blocked the binding of alpha 4 to DNA, one (H950) reacted with oligopeptide no. 3 near the N terminal of the protein, whereas the second (H942) reacted with oligopeptide no. 23 near the C terminus of the 288-amino-acid polypeptide. In further tests, oligopeptide no. 19 was found to compete with two host proteins, designated as alpha H1 and alpha H2-alpha H3, for binding to DNA as well as to retard DNA in a band shift assay, whereas oligopeptides no. 26, 27, and 28 enhanced the binding of alpha 4 to DNA. Moreover, oligopeptide no. 27 was also found to retard DNA in a band shift assay. Polypeptide no. 19 competed with alpha 4 for binding to DNA, whereas no. 27 neither enhanced nor competed with the binding of the host polypeptide alpha H1 to its binding site in the promoter-regulatory domain of an alpha gene, but did enhance the binding of the alpha H2-alpha H3 protein to its binding site. In contrast to these results, the truncated alpha 4 polypeptide, 825 amino acids long, bound to the viral DNA, whereas a shorter, 519-amino-acid-long, truncated polypeptide did not. The 825-amino-acid polypeptide was previously shown to induce in transient expression of a late (gamma 2) viral gene.     

Differential affinity and cooperativity functions of the amino-terminal 70 residues of lambda integrase

The site-specific recombinase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein that binds two different classes of DNA-binding sites within its recombination target sites. The several functions of Int are apportioned between a large carboxy-terminal domain that cleaves and ligates DNA at each of its four "core-type" DNA-binding sites and a small amino-terminal domain, whose primary function is binding to each of its five "arm-type" DNA sites, which are distant from the core region. Int bridges between the two classes of binding sites are facilitated by accessory DNA-bending proteins that along with Int comprise higher-order recombinogenic complexes. We show here that although the 64 amino-terminal residues of Int bind efficiently to a single arm site, this protein cannot form doubly bound complexes on adjacent arm sites. However, 1-70 Int does show the same cooperative binding to adjacent arm sites as the full length protein. We also found that 1-70 Int specifies cooperative interactions with the accessory protein Xis when the two are bound to their adjacent cognate sites P2 and X1, respectively. To complement the finding that these two amino-terminal domain functions (along with arm DNA binding) are all specified by residues 1-70, we determined that Thr75 is the first residue of the minimal carboxy-terminal domain, thereby identifying a specific interdomain linker region. We have measured the affinity constants for Int binding to each of the five arm sites and the cooperativity factors for Int binding to the two pairs of adjacent arm sites, and we have identified several DNA structural features that contribute to the observed patterns of Int binding to arm sites. Taken together, the results highlight several interesting features of arm DNA binding that invite speculation about additional levels of complexity in the regulation of lambda site-specific recombination.     

Nucleosome linker DNA contacts and induces specific folding of the intrinsically disordered H1 carboxyl-terminal domain

Linker histones play essential roles in the chromatin structure of higher eukaryotes. While binding to the surface of nucleosomes is directed by an ～ 80-amino-acid-residue globular domain, the structure and interactions of the lysine-rich ～ 100-residue C-terminal domain (CTD), primarily responsible for the chromatin-condensing functions of linker histones, are poorly understood. By quantitatively analyzing binding of a set of H1 CTD deletion mutants to nucleosomes containing various lengths of linker DNA, we have identified interactions between distinct regions of the CTD and nucleosome linker DNA at least 21 bp from the edge of the nucleosome core. Importantly, partial CTD truncations caused increases in H1 binding affinity, suggesting that significant entropic costs are incurred upon binding due to CTD folding. van't Hoff entropy/enthalpy analysis and intramolecular fluorescent resonance energy transfer (FRET) studies indicate that the CTD undergoes substantial nucleosome-directed folding, in a manner that is distinct from that which occurs upon H1 binding to naked DNA. In addition to defining critical interactions between the H1 CTD and linker DNA, our data indicate that the H1 CTD is an intrinsically disordered domain and provide important insights into the biological function of this protein.     

Identification of the substrate binding site in the N-terminal TBP-like domain of RNase H3

Ribonuclease H3 from Bacillus stearothermophilus (Bst-RNase H3) has the N-terminal TBP-like substrate-binding domain. To identify the substrate binding site in this domain, the mutant proteins of the intact protein and isolated N-domain, in which six of the seventeen residues corresponding to those involved in DNA binding of TBP are individually mutated to Ala, were constructed. All of them exhibited decreased enzymatic activities and/or substrate-binding affinities when compared to those of the parent proteins, suggesting that the N-terminal domain of RNase H3 uses the flat surface of the β-sheet for substrate binding as TBP to bind DNA. This domain may greatly change conformation upon substrate binding.     

The maize ID1 flowering time regulator is a zinc finger protein with novel DNA binding properties

The INDETERMINATE protein, ID1, plays a key role in regulating the transition to flowering in maize. ID1 is the founding member of a plant-specific zinc finger protein family that is defined by a highly conserved amino sequence called the ID domain. The ID domain includes a cluster of three different types of zinc fingers separated from a fourth C2H2 finger by a long spacer; ID1 is distinct from other ID domain proteins by having a much longer spacer. In vitro DNA selection and amplification binding assays and DNA binding experiments showed that ID1 binds selectively to an 11 bp consensus motif via the ID domain. Unexpectedly, site-directed mutagenesis of the ID1 protein showed that zinc fingers located at each end of the ID domain are not required for binding to the consensus motif despite the fact that one of these zinc fingers is a canonical C2H2 DNA binding domain. In addition, an ID1 in vitro deletion mutant that lacks the extra spacer between zinc fingers binds the same 11 bp motif as normal ID1, suggesting that all ID domain-containing proteins recognize the same DNA target sequence. Our results demonstrate that maize ID1 and ID domain proteins have novel zinc finger configurations with unique DNA binding properties.     

MeCP2 preferentially binds to methylated linker DNA in the absence of the terminal tail of histone H3 and independently of histone acetylation

Methyl CpG binding protein 2 (MeCP2) is a basic protein that contains a DNA methyl binding domain. The mechanism by which the highly positive charge of MeCP2 and its ability to bind methylated DNA contribute to the specificity of its binding to chromatin has long remained elusive. In this paper, we show that MeCP2 binds to nucleosomes in a very similar way to linker histones both in vitro and in vivo. However, its binding specificity strongly depends on DNA methylation. We also observed that as with linker histones, this binding is independent of the core histone H3 N-terminal tail and is not affected by histone acetylation.     

Solution structure and characterization of the DNA-binding activity of the B3BP-Smr domain

The MutS1 protein recognizes unpaired bases and initiates mismatch repair, which are essential for high-fidelity DNA replication. The homologous MutS2 protein does not contribute to mismatch repair, but suppresses homologous recombination. MutS2 lacks the damage-recognition domain of MutS1, but contains an additional C-terminal extension: the small MutS-related (Smr) domain. This domain, which is present in both prokaryotes and eukaryotes, has previously been reported to bind to DNA and to possess nicking endonuclease activity. We determine here the solution structure of the functionally active Smr domain of the Bcl3-binding protein (also known as Nedd4-binding protein 2), a protein with unknown function that lacks other domains present in MutS proteins. The Smr domain adopts a two-layer α-β sandwich fold, which has a structural similarity to the C-terminal domain of IF3, the R3H domain, and the N-terminal domain of DNase I. The most conserved residues are located in three loops that form a contiguous, exposed, and positively charged surface with distinct sequence identity for prokaryotic and eukaryotic Smr domains. NMR titration experiments and DNA binding studies using Bcl3-binding protein-Smr domain mutants suggested that these most conserved loop regions participate in DNA binding to single-stranded/double-stranded DNA junctions. Based on the observed DNA-binding-induced multimerization, the structural similarity with both subdomains of DNase I, and the experimentally identified DNA-binding surface, we propose a model for DNA recognition by the Smr domain.     

Inner nuclear membrane protein LBR preferentially interacts with DNA secondary structures and nucleosomal linker

The lamin B receptor (LBR) is an integral protein of inner nuclear membrane whose nucleoplasmic amino-terminal domain contributes to the attachment of the membrane to chromatin. Here we analyzed the interactions of a recombinant GST protein containing the amino-terminal domain of the protein with in vitro reconstituted nucleosomes and short DNA fragments. Data show that the LBR amino-terminal domain (AT) binds linker DNA but does not interact with the nucleosome core. Titration and competition studies revealed that the interaction between LBR AT and DNA is saturable, of high affinity (K(D) approximately 4 nM), independent of DNA sequence, and enhanced by DNA curvature and supercoiling. In this respect, LBR amino-terminal domain binding to nucleosomes is similar to that of histone H1 and non histone proteins HMG1/2 which both bind preferentially to linker DNA and present a significant affinity for DNA secondary structures.     

Additional binding sites for the pyruvate dehydrogenase kinase but not for protein X in the assembled core of the mammalian pyruvate dehydrogenase complex: binding region for the kinase.

A standard resolution of the bovine kidney pyruvate dehydrogenase complex yields a subcomplex composed of approximately 60 dihydrolipoyl transacetylase (E2) subunits, approximately 6 protein X subunits, and approximately 2 pyruvate dehydrogenase kinase heterodimers (KcKb). Using a preparation of resolved kinase in which Kc much greater than Kb, E2-X-KcKb subcomplex additionally bound at least 15 catalytic subunits of the kinase (Kc) and a much lower level of Kb. The binding of Kc to E2 greatly enhanced kinase activity even at high levels of bound kinase. Free protein X, functional in binding the E3 component, did not bind to E2-X-KcKb subcomplex. This pattern of binding Kc but not protein X was unchanged either with a preparation of E2 oligomer greatly reduced in protein X or with subcomplex from which the lipoyl domain of protein X was selectively removed. The bound inner domain of protein X associated with the latter subcomplex did not exchange with free protein X. These data support the conclusion that E2 subunits bind the Kc subunit of the kinase and suggest that the binding of the inner domain of protein X to the inner domain of the transacetylase occurs during the assembly of the oligomeric core. Selective release of a fragment of E2 subunits that contain the lipoyl domains (E2L fragment) releases the kinase (M. Rahmatullah et al., 1990, J. Biol. Chem. 265, 14,512-14,517). Sucrose gradient centrifugation yielded an E2L-kinase fraction with an increased ratio of the kinase to E2L fragment. A monoclonal antibody specific for E2L was attached to a gel matrix. Binding of E2L fragment also led to specific binding of the kinase. Extensive washing did not reduce the level of bound kinase. Thus, the kinase is tightly bound by the lipoyl domain region of E2.