Acidic Tail Research Articles

Biochemical Properties of Single-Stranded DNA-Binding Protein from Mycobacterium smegmatis, a Fast-growing Mycobacterium and Its Physical and Functional Interaction with Uracil DNA Glycosylases

The single-stranded DNA-binding proteins (SSBs) are vital to virtually all DNA functions. Here, we report on the biochemical properties of SSB from a fast-growing mycobacteria, Mycobacterium smegmatis, and the interaction of the homotetrameric SSBs with uracil DNA glycosylases (UDGs) from M.smegmatis (Msm), Mycobacterium tuberculosis (Mtu) and Escherichia coli (Eco). UDG is a crucial DNA repair enzyme, which removes the promutagenic uracil residues. MsmSSB stimulates activity of the homologous MsmUDG and of the heterologous Mtu-, and Eco-UDGs. On the contrary, while the MtuSSB stimulates the MtuUDG, it inhibits the other two UDGs. Although the MsmSSB shares 84% identity with MtuSSB, the two are strikingly different, in that MsmSSB contains a glycine-rich segment (11 out of 13 residues) in the spacer connecting the N-terminal DNA-binding domain with the C-terminal acidic tail. While the DNA-binding properties of MsmSSB, such as its affinity to oligomeric DNA, requirement of minimum size DNA and the modes of interaction are indistinguishable from those of Eco-, and Mtu-SSBs, it is unclear if the glycine-rich segment confers structural advantage to MsmSSB, responsible for its stimulatory effect on all UDGs tested. More importantly, by using a small polypeptide inhibitor of UDGs, and the deletion mutants of SSBs, we suggest that the C-terminal acidic tail of the SSBs interacts within the DNA-binding groove of the UDGs, and propose a role for SSBs in the recruitment of UDGs to the damaged DNA.

Stress-Induced Aggregation Profiles of GST−α-Synuclein Fusion Proteins: Role of the C-Terminal Acidic Tail of α-Synuclein in Protein Thermosolubility and Stability

Alpha-synuclein is a well-known heat-resistant protein that does not aggregate upon heat treatment, whereas glutathione S-transferase (GST) is a heat-labile protein that easily precipitates as a result of thermal stress. This paper reports the role of the C-terminal acidic tail of alpha-synuclein in protein thermosolubility and stability. The region of alpha-synuclein that is responsible for the heat resistance was initially investigated using a series of deletion mutants, and the C-terminal acidic tail (residues 96-140) was found to be crucial for the thermosolubility of alpha-synuclein. The thermal behavior of the GST-alpha-synuclein fusion protein was next investigated, and the fusion protein was seen to be extremely heat-resistant. Using a series of GST-synuclein deletion mutants, the C-terminal acidic tail of alpha-synuclein was shown to play a critical role in conferring the heat resistance of the fusion proteins. Furthermore, the acidic tail appeared to protect the fusion protein from pH- and metal-induced protein aggregation, suggesting that the acidic tail can increase the virtual stability of the protein by protecting it from the aggregation induced by environmental stresses. Interestingly, the acidic tail also appeared to protect the GST enzyme from the thermal inactivation to a considerable extent. However, CD analysis of the heat-induced secondary structural changes of the GST-alpha-synuclein fusion protein revealed that the fusion protein is irreversibly denatured by heat treatment with a slightly lowered melting temperature (Tm). Thus, the results demonstrate that introducing an acidic tail into GST promotes the thermosolubility and virtual stability of the fusion protein, although it might be unfavorable for its intrinsic stability.

Sequencing of intron 3 of HMGA2 uncovers the existence of a novel exon.

Aberrations affecting the gene encoding the high mobility group protein HMGA2 (formerly HMGIC) have been found in a variety of human tumors, e.g., uterine leiomyomas, lipomas, and pulmonary chondroid hamartomas. These aberrations lead to fusion genes, transcriptional up-regulation, or aberrant transcripts of HMGA2. In the latter case, truncated transcripts consisting of exons 1 to 3 of HMGA2, encoding the three DNA-binding domains, and ectopic sequences derived from chromosome 12 are frequent. There are several lines of evidence indicating that the biological and tumorigenic features of truncated HMGA2 derivatives, i.e., those composed of the DNA-binding domains and a shortened acidic tail, clearly differ from those of the normal protein consisting of three DNA-binding domains and one large acidic tail. By sequencing the complete 112 kb third intron of HMGA2, we were able to detect several of the ectopic sequences, known as fused to HMGA2. Expression studies revealed co-expression of one of these transcripts with the normal transcript in tumors with 12q14-15 aberrations as well as in other tumors, and in normal tissues. Thus, this transcript (HMGA2b) is flanked by an alternative terminal exon of HMGA2. Due to the loss of the part encoding the acidic tail, the expression of the latter transcript may have more striking effects than the "wild type" HMGA2 (HMGA2a) in terms of tumorigenesis. This finding clearly indicates that functional studies also should address the role of the HMGA2b transcript.

The X-ray Crystal Structure and Putative Ligand-derived Peptide Binding Properties of γ-Aminobutyric Acid Receptor Type A Receptor-associated Protein

The gamma-aminobutyric acid receptor type A (GABA(A)) receptor-associated protein (GABARAP) has been reported to mediate the interaction between the GABA(A) receptor and microtubules. We present the three-dimensional structure of GABARAP obtained by x-ray diffraction at 1.75 A resolution. The structure was determined by molecular replacement using the structure of the homologous protein GATE-16. NMR spectroscopy of isotope-labeled GABARAP showed the structure in solution to be compatible with the overall fold but showed evidence of conformation heterogeneity that is not apparent in the crystal structure. We assessed the binding of GABARAP to peptides derived from reported binding partner proteins, including the M3-M4 loop of the gamma2 subunit of the GABA(A) receptor and the acidic carboxyl-terminal tails of human alpha- and beta-tubulin. There is a small area of concentrated positive charge on one surface of GABARAP, which we found interacts weakly with all peptides tested, but we found no evidence for specific binding to the proposed physiological target peptides. These results are compatible with a more general role in membrane targeting and transportation for the GABARAP family of proteins.

XKCM1 acts on a single protofilament and requires the C terminus of tubulin

The stability of microtubules during the cell-cycle is regulated by a number of cellular factors, some of which stabilize microtubules and others that promote breakdown. XKCM1 is a kinesin-like protein that induces microtubule depolymerization and is required for mitotic spindle assembly. We have examined the binding and depolymerization effects of XKCM1 on different tubulin polymers in order to learn about its mechanism of action. Zinc-induced tubulin polymers, characterized by an anti-parallel protofilament arrangement, are depolymerized by XKCM1, indicating that this enzyme acts on a single protofilament. GDP-tubulin rings, which correspond to the low-energy state of tubulin, are stable only under conditions that inhibit XKCM1 depolymerizing activity, but can be stabilized by XKCM1 bound to AMPPNP. Tubulin polymers made of subtilisin-treated tubulin (lacking the tubulin C-terminal tail) are resistant to XKCM1-induced depolymerization, suggesting that the interaction of the acidic tail of tubulin with basic residues in XKCM1 unique to Kin I proteins is required for depolymerization.

Functional analysis of rat acidic calponin.

Recombinant acidic calponin, a member of the calponin family, interacted with F-actin, but not with microtubules, desmin filaments, tropomyosin, calmodulin, S100 and phosphatidylserine (PS) vesicles with significant affinity. The bindings of acidic calponin to F-actin occurred in a concentration-dependent manner and were saturated at a molar ratio of about 1 acidic calponin to 1-2 actin molecules. The apparent Kd value of acidic calponin to F-actin was calculated to be 1.6 x 10(5) M(-1). Chemical cross-linking experiments indicated that a 1:1 molar covalent complex of acidic calponin and actin monomer was produced as in the case of basic calponinactin binding. No significant morphologic change of F-actin was observed by the addition of acidic calponin. Acidic calponin had little effect on actomyosin Mg2+-ATPase activity unlike basic calponin. Basic calponin partially competed with acidic calponin for binding to F-actin. Domain mapping with V8 protease revealed that acidic calponin binding site resided within the C-terminal 16 kDa fragment of actin, where the binding of basic calponin also occurs. However, both calponins showed reversal effects on fluorescence intensity of pyrene-labeled F-actin. Fragments of acidic calponin with 30 and 22 kDa, lacking the C-terminal acidic tail, were bound to F-actin. Interestingly, both the fragments became bound to PS vesicles, but not to other components. Circular dichroism studies showed that limited digestion of acidic calponin resulted in about 30% decrease of alpha-helix and beta contents. The present results suggest that acidic calponin is functionally distinct from basic calponin and expresses a novel characteristic after removal of the acidic tail region.

Conformational stability of six truncated cHMG1a proteins studied in their mixture by H/D exchange and electrospray ionization mass spectrometry.

The high mobility group (HMG) proteins are abundant non-histone components of eukaryotic chromatin. The presence of C-terminal acidic tails is a common feature of the majority of HMG proteins. Although the biological significance of the acidic domains is not clear, they are conferring conformational and metabolic stability to the proteins in vitro. Moreover, the length and net charge of the acidic tails affect the strength of HMG protein interaction with DNA. Synthesis of an insect HMG protein by standard recombinant technology in bacteria leads to a mixture of the intact protein (cHMG1a-(1-113) (I)) and a series of its degradation products truncated at the C tail: cHMG1a-(1-111) (II); cHMG1a-(1-110) (III); cHuMGla-(1-109) (IV); cHMG1a-(1-108) (V); cHMG1a-(1-107) (VI); cHMG1a-(1-106) (VII). The proteins differ from each other only by the number of amino-acid residues at the C-terminal tail. We used H/D exchange mass spectrometry to characterize the stability of the proteins directly in their mixture. The results show that the proteins I-V and VII have very similar conformations. The protein VI is less compact and exchanges its protons faster than the others. It may be concluded that the C-terminal tail influences the conformation of the cHMG1a protein and that individual residues in this part of the protein play a key role in its compactness.

Refined structure of αβ-tubulin at 3.5 Å resolution

We present a refined model of the αβ-tubulin dimer to 3.5 Å resolution. An improved experimental density for the zinc-induced tubulin sheets was obtained by adding 114 electron diffraction patterns at 40-60 ° tilt and increasing the completeness of structure factor amplitudes to 84.7 %. The refined structure was obtained using maximum-likelihood including phase information from experimental images, and simulated annealing Cartesian refinement to an R-factor of 23.2 and free R-factor of 29.7. The current model includes residues α:2-34, α:61-439, β:2-437, one molecule of GTP, one of GDP, and one of taxol, as well as one magnesium ion at the non-exchangeable nucleotide site, and one putative zinc ion near the M-loop in the α-tubulin subunit. The acidic C-terminal tails could not be traced accurately, neither could the N-terminal loop including residues 35-60 in the α-subunit. There are no major changes in the overall fold of tubulin with respect to the previous structure, testifying to the quality of the initial experimental phases. The overall geometry of the model is, however, greatly improved, and the position of side-chains, especially those of exposed polar/charged groups, is much better defined. Three short protein sequence frame shifts were detected with respect to the non-refined structure. In light of the new model we discuss details of the tubulin structure such as nucleotide and taxol binding sites, lateral contacts in zinc-sheets, and the significance of the location of highly conserved residues.

Role of the high mobility group A proteins in human lipomas.

The HMGA family is comprised of four proteins: HMGA1a, HMGA1b, HMGA1c and HMGA2. The first three proteins are products of the same gene, HMGA1, generated through an alternative splicing mechanism. The HMGA proteins are involved in the regulation of chromatin structure and HMGA DNA-binding sites have been identified in functional regions of many gene promoters. Rearrangements of the HMGA2 gene have been frequently detected in human benign tumors of mesenchymal origin including lipomas. 12q13-15 chromosomal translocations involving the HMGA2 gene locus, account for these rearrangements. The HMGA proteins have three AT-hook domains and an acidic C-terminal tail. The HMGA2 modifications consist in the loss of the C-terminal tail and fusion with ectopic sequences. A pivotal role of the HMGA2 rearrangements in the process of lipomagenesis is suggested by experiments showing that transgenic mice carrying a truncated HMGA2 gene showed a giant phenotype together with abdominal/pelvic lipomatosis. As HMGA2 null mice showed a great reduction in fat tissue, a positive role of the HMGA2 gene in adipocytic cell proliferation is proposed. More recently, similar alterations of the HMGA1 gene have been described. As the block of the HMGA1 protein synthesis induces an increase in growth rate of the pre-adipocytic cell line 3T3-L1, we suggest a negative role of the HMGA1 proteins in adipocytic cell growth and, therefore, we propose that adipocytic cell growth derives from the balance of the HMGA1 and HMGA2 protein functions.

Granzyme A activates an endoplasmic reticulum-associated caspase-independent nuclease to induce single-stranded DNA nicks.

The cytotoxic T lymphocyte protease granzyme A (GzmA) initiates a novel caspase-independent cell death pathway characterized by single-stranded DNA nicking. The previously identified GzmA substrate SET is in a multimeric 270-420-kDa endoplasmic reticulum-associated complex that also contains the tumor suppressor protein pp32. GzmA cleaved the nucleosome assembly protein SET after Lys(176) and disrupted its nucleosome assembly activity. The purified SET complex required only GzmA to reconstitute single-stranded DNA nicking in isolated nuclei. DNA nicking occurred independently of caspase activation. The SET complex contains a 25-kDa Mg(2+)-dependent nuclease that degrades calf thymus DNA and plasmid DNA. Thus, GzmA activates a DNase (GzmA-activated DNase) within the SET complex to produce a novel form of DNA damage during cytotoxic T lymphocyte-mediated death.

HPop5, a Protein Subunit of the Human RNase MRP and RNase P Endoribonucleases

The RNase MRP and RNase P particles both function as endoribonucleases. RNase MRP has been implicated in the processing of precursor-rRNA, whereas RNase P has been shown to function in the processing of pre-tRNA. Both ribonucleoprotein particles have an RNA component that can be folded into a similar secondary structure and share several protein components. We have identified human, rat, mouse, cow, and Drosophila homologues of the Pop5p protein subunit of the yeast RNase MRP and RNase P complexes. The human Pop5 cDNA encodes a protein of 163 amino acids with a predicted molecular mass of 18.8 kDa. Polyclonal antibodies raised against recombinant hPop5 identified a 19-kDa polypeptide in HeLa cells and showed that hPop5 is associated with both RNase MRP and RNase P. Using affinity-purified anti-hPop5 antibodies, we demonstrated that the endogenous hPop5 protein is localized in the nucleus and accumulates in the nucleolus, which is consistent with its association with RNase MRP and RNase P. Catalytically active RNase P was partially purified from HeLa cells, and hPop5 was shown to be associated with it. Finally, the evolutionarily conserved acidic C-terminal tail of hPop5 appeared to be required neither for complex formation nor for RNase P activity.

HMG1 and 2: architectural DNA-binding proteins.

HMG1 and 2 (high mobility group proteins 1 and 2; renamed HMGB1 and 2) contain two DNA-binding HMG-box domains (A and B) and a long acidic C-terminal domain. They bind DNA without sequence specificity, but have a high affinity for bent or distorted DNA, and bend linear DNA. The individual A and B boxes (which, although broadly similar, show both structural and functional differences) exhibit many of the structure-specific properties of the whole protein. The acidic tail modulates the affinity of the tandem HMG boxes in HMG1 and 2 for a variety of DNA targets, including four-way junctions, but not distorted DNA minicircles, to which the proteins bind with very high affinity. HMG1 and 2 appear to play important architectural roles in the assembly of nucleoprotein complexes in a variety of biological processes, for example V(D)J recombination, the initiation of transcription, and DNA repair.

The Basal Transcription Factors TBP and TFB from the Mesophilic Archaeon Methanosarcina mazeii: Structure and Conformational Changes upon Interaction with Stress-gene Promoters

Transcription of archaeal non-stress genes involves the basal factors TBP and TFB, homologs of the eucaryal TATA-binding protein and transcription factor IIB, respectively. No comparable information exists for the archaeal molecular-chaperone, stress genes hsp70(dnaK), hsp40(dnaJ), and grpE. These do not occur in some archaeal species, but are present in others possibly due to lateral transfer from bacteria, which provides a unique opportunity to study regulation of stress-inducible bacterial genes in organisms with eukaryotic-like transcription machinery. Among the Archaea with the genes, those from the mesophilic methanogen Methanosarcina mazeii are the only ones whose basal (constitutive) and stress-induced transcription patterns have been determined. To continue this work, tbp and tfb were cloned from M. mazeii , sequenced, and the encoded recombinant proteins characterized in solution, separately and in complex with each other and with DNA. M. mazeii TBP ranks among the shortest within Archaea and, contrary to other archaeal TBPs, it lacks tryptophan or an acidic tail at the C terminus and has a basic N-terminal third. M. mazeii TFB is similar in length to archaeal and eucaryal homologs and all have a zinc finger and HTH motifs. Phylogenetically, the archaeal and eucaryal proteins form separate clusters and the M. mazeii molecules are closer to the homologs from Archaeoglobus fulgidus than to any other. Antigenically, M. mazeii TBP and TFB are close to archaeal homologs within each factor family, but the two families are unrelated. The purified recombinant factors were functionally active in a cell-free in vitro transcription system, and were interchangeable with the homologs from Methanococcus thermolithotrophicus. The M. mazeii factors have a similar secondary structure by circular dichroism (CD). The CD spectra changed upon binding to the promoters of the stress genes grpE, dnaK, and dnaJ, with the changes being distinctive for each promoter; in contrast, no effect was produced by the promoter of a non-stress-gene. Factor(s)-DNA modeling predicted that modifications of H bonds are caused by TBP binding, and that these modifications are distinctive for each promoter. It also showed which amino acid residues would contact an extended TATA box with a B recognition element, and evolutionary conservation of the TBP-TFB-DNA complex orientation between two archaeal organisms with widely different optimal temperature for growth (37 and 100 °C).

Structural requirements for cooperative binding of HMG1 to DNA minicircles

DNA minicircles, where the length of DNA is below the persistence length, are highly effective, preferred, ligands for HMG-box proteins. The proteins bind to them “structure-specifically” with affinities in the nanomolar range, presumably to an exposed widened minor groove. To understand better the basis of this preference, we have studied the binding of HMG1 (which has two tandem HMG boxes linked by a basic extension to a long acidic tail) and Drosophila HMG-D (one HMG box linked by a basic region to a short and less acidic tail), and their HMG-box domains, to 88 bp and 75 bp DNA minicircles. In some cases we see cooperative binding of two molecules to the circles. The requirements for strong cooperativity are two HMG boxes and the basic extension; the latter also appears to stabilize and constrain the complex, preventing binding of further protein molecules. HMG-D, with a single HMG box, does not bind cooperatively. In the case of HMG1, the acidic tail is not required for cooperativity and does not affect binding significantly, in contrast to a much greater effect with linear DNA, or even four-way junctions (another distorted DNA substrate). Such effects could be relevant in the hierarchy of binding of HMG-box proteins to DNA distortions in vivo, where both single-box and two-box proteins might co-exist, with or without basic extensions and acidic tails.

Microsporidian invasion apparatus: identification of a novel polar tube protein and evidence for clustering of ptp1 and ptp2 genes in three Encephalitozoon species.

Microsporidia are unicellular eukaryotes occurring as obligate intracellular parasites which produce resistant spores. A unique motile process is represented by the sudden extrusion of the sporal polar tube for initiating entry of the parasite into a new host cell. The complete sequence of an acidic proline-rich polar tube protein (renamed PTP1) has been previously reported for Encephalitozoon cuniculi and E. hellem. Our immunological investigations provided evidence for an additional PTP in E. cuniculi, termed PTP2. The corresponding gene was sequenced and then expressed in Escherichia coli. As expected, mouse antibodies raised against the recombinant protein reacted specifically with the polar tube. The single copy ptp1 and ptp2 genes of E. cuniculi were tandemly arranged on chromosome VI. Polyadenylation of the mRNAs was demonstrated. Identification and sequencing of homologous genes in the two other human-infecting Encephalitozoon species (ptp2 in E. hellem and ptp1 and ptp2 in E. intestinalis) were facilitated by conserved gene clustering. PTP2 appears as a novel structural protein (30 kDa) with a basic lysine-rich core and an acidic tail. Unlike PTP1, this protein is devoid of large tandem repeats. The interspecies conservation of cysteine residues supports a major role of disulfide bridges in polar tube assembly. The two PTPs should serve as both molecular markers of spore differentiation and diagnostic tools.

The effect of the acidic tail on the DNA-binding properties of the HMG1,2 class of proteins: insights from tail switching and tail removal

The high-mobility group (HMG) proteins HMG1, HMG2 and HMG2a are relatively abundant vertebrate DNA-binding and bending proteins that bind with structure specificity, rather than sequence specificity, and appear to play an architectural role in the assembly of nucleoprotein complexes. They have two homologous “HMG-box” DNA-binding domains (which show about 80 % homology) connected by a short basic linker to an acidic carboxy-terminal tail that differs in length between HMG1 and 2. To gain insights into the role of the acidic tail, we examined the DNA-binding properties of HMG1, HMG2b and HMG2a from chicken erythrocytes (corresponding to HMG1, HMG2 and HMG2a in other vertebrates). HMG1, with the longest acidic tail, is less effective than HMG2a and 2b (at a given molar input ratio) in supercoiling relaxed, closed circular DNA, in inducing ligase-mediated circularisation of an 88 bp DNA fragment, and in binding to four-way DNA junctions in a gel-shift assay. Removal of the acidic tail increases the affinity of the HMG boxes for DNA and largely abolishes the differences between the three species. Switching the acidic tail of HMG1 for that of HMG2a or 2b gives hybrid proteins with essentially the same DNA-binding properties as HMG2a, 2b. The length (and possibly sequence) of the acidic tail thus appears to be the dominant factor in mediating the differences in properties between HMG1, 2a and 2b and finely tunes the rather similar DNA-binding properties of the tandem HMG boxes, presumably to fulfill different cellular roles. The tail is essential for structure-selective DNA-binding of the HMG boxes to DNA minicircles in the presence of equimolar linear DNA, and has little effect on the affinity for this already highly distorted DNA ligand, in contrast to binding to linear and four-way junction DNA.

The carboxy terminus of Tub4p is required for gamma-tubulin function in budding yeast.

The role of gamma-tubulin in microtubule nucleation is well established, however, its function in other aspects of microtubule organization is unknown. The carboxy termini of alpha/beta-tubulins influence the assembly and stability of microtubules. We investigated the role of the carboxy terminus of yeast gamma-tubulin (Tub4p) in microtubule organization. This region consists of a conserved domain (DSYLD), and acidic tail. Cells expressing truncations lacking the DSYLD domain, tail or both regions are temperature sensitive for growth. Growth defects of tub4 mutants lacking either or both carboxy-terminal domains are suppressed by the microtubule destabilizing drug benomyl. tub4 carboxy-terminal mutants arrest as large budded cells with short bipolar spindles positioned at the bud neck. Electron microscopic analysis of wild-type and CTR mutant cells reveals that SPBs are tightly associated with the bud neck/cortex by cytoplasmic microtubules in mutants lacking the tail region (tub4-delta 444, tub4-delta 448). Mutants lacking the DSYLD residues (tub4-delta 444, tub4-delta DSYLD) form many cytoplasmic microtubules. We propose that the carboxy terminus of Tub4p is required for re-organization of the microtubules upon completion of nuclear migration, and facilitates spindle elongation into the bud.

Protein ligands to HuR modulate its interaction with target mRNAs in vivo.

AU-rich elements (AREs) present in the 3' untranslated regions of many protooncogene, cytokine, and lymphokine messages target them for rapid degradation. HuR, a ubiquitously expressed member of the ELAV (embryonic lethal abnormal vision) family of RNA binding proteins, selectively binds AREs and stabilizes ARE-containing mRNAs in transiently transfected cells. Here, we identify four mammalian proteins that bind regions of HuR known to be essential for its ability to shuttle between the nucleus and the cytoplasm and to stabilize mRNA: SETalpha, SETbeta, pp32, and acidic protein rich in leucine (APRIL). Three have been reported to be protein phosphatase 2A inhibitors. All four ligands contain long, acidic COOH-terminal tails, while pp32 and APRIL share a second motif: rev-like leucine-rich repeats in their NH(2)-terminal regions. We show that pp32 and APRIL are nucleocytoplasmic shuttling proteins that interact with the nuclear export factor CRM1 (chromosomal region maintenance protein 1). The inhibition of CRM1 by leptomycin B leads to the nuclear retention of pp32 and APRIL, their increased association with HuR, and an increase in HuR's association with nuclear poly(A)+ RNA. Furthermore, transcripts from the ARE-containing c-fos gene are selectively retained in the nucleus, while the cytoplasmic distribution of total poly(A)+ RNA is not altered. These data provide evidence that interaction of its ligands with HuR modulate HuR's ability to bind its target mRNAs in vivo and suggest that CRM1 is instrumental in the export of at least some cellular mRNAs under certain conditions. We discuss the possible role of these ligands upstream of HuR in pathways that govern the stability of ARE-containing mRNAs.

Identification of Residues within Two Regions Involved in Self-association of Viral Histone-like Protein p6 from Phage Ø29

Protein p6 of Bacillus subtilis phage theta29 is involved in the initiation of viral DNA replication and transcription by forming a multimeric nucleoprotein complex with the phage DNA. Based on this, together with its abundance and its capacity to bind to the whole viral genome, it has been proposed to be a viral histone-like protein. Protein p6 is in a monomer-dimer-oligomer equilibrium association. We have identified protein p6 mutants deficient in self-association by testing random mutants obtained by degenerated polymerase chain reaction in an in vivo assay for dimer formation. The mutations were mainly clustered in two regions located at the N terminus, and the central part of the protein. Site-directed single mutants, corresponding to those found in vivo, have been constructed and purified. Mutant p6A44V, located at the central part of the protein, showed an impaired dimer formation ability, and a reduced capacity to bind DNA and to activate the initiation of O29 DNA replication. Mutant p6I8T has at least 10-fold reduced self-association capacity, does not bind DNA nor activate O29 DNA initiation of replication. C-terminal deletion mutants showed an enhanced dimer formation capacity. The highly acidic tail, removed in these mutants, is proposed to modulate the protein p6 self-association.

HMG1 protein stimulates DNA end joining by promoting association of DNA molecules via their ends.

High mobility group (HMG) 1 protein is a highly abundant and an evolutionarily conserved chromosomal protein with two homologous DNA-binding domains (HMG boxes), A and B, attached by a short basic region to an acidic C-terminal tail. The protein has been implicated in a number of fundamental biological processes including DNA replication, transcription, recombination and repair. We demonstrate that HMG1 is able to enhance cohesive-end and blunt-end DNA ligation by T4 DNA ligase via its B domain. The C-terminal flanking sequence of the B domain (seven basic residues out of approximately 18) and a number of conserved amino-acid residues within the HMG box (mainly basic or hydrophobic) are required for efficient stimulation of ligation. Pull-down assays, electron and scanning force microscopy revealed that HMG1 can associate two DNA molecules via their ends even in the absence of complementary overhangs. We propose that HMG1 protein may be involved in the rejoining of DNA breaks by different DNA ligases due to its ability to bring DNA duplexes and their termini into a close proximity while leaving the ends accessible for ligation.