Replication protein A. Characterization and crystallization of the DNA binding domain

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein in eukaryotic cells. The DNA binding activity of human RPA has been previously localized to the N-terminal 441 amino acids of the 70-kDa subunit, RPA70. We have used a combination of limited proteolysis and mutational analysis to define the smallest soluble fragment of human RPA70 that retains complete DNA binding activity. This fragment comprises residues 181-422. RPA181-422 bound DNA with the same affinity as the 1-441 fragment and had a DNA binding site of 8 nucleotides or less. RPA70 fragments were subjected to crystal trials in the presence of single-stranded DNA, and diffraction quality crystals were obtained for RPA181-422 bound to octadeoxycytidine. The RPA181-422 co-crystals belonged to the P2(1)2(1)2(1) space group, with unit cell dimensions of a = 34.3 A, b = 78.0 A, and c = 95.4 A and diffracted to a resolution of 2.1 A.     

DNA annealing by RAD52 protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA

Homologous recombination in Saccharomyces cerevisiae depends critically on RAD52 function. In vitro, Rad52 protein preferentially binds single-stranded DNA (ssDNA), mediates annealing of complementary ssDNA, and stimulates Rad51 protein-mediated DNA strand exchange. Replication protein A (RPA) is a ssDNA-binding protein that is also crucial to the recombination process. Herein we report that Rad52 protein effects the annealing of RPA-ssDNA complexes, complexes that are otherwise unable to anneal. The ability of Rad52 protein to promote annealing depends on both the type of ssDNA substrate and ssDNA binding protein. RPA allows, but slows, Rad52 protein-mediated annealing of oligonucleotides. In contrast, RPA is almost essential for annealing of longer plasmid-sized DNA but has little effect on the annealing of poly(dT) and poly(dA), which are relatively long DNA molecules free of secondary structure. These results suggest that one role of RPA in Rad52 protein-mediated annealing is the elimination of DNA secondary structure. However, neither Escherichia coli ssDNA binding protein nor human RPA can substitute in this reaction, indicating that RPA has a second role in this process, a role that requires specific RPA-Rad52 protein interactions. This idea is confirmed by the finding that RPA, which is complexed with nonhomologous ssDNA, inhibits annealing but the human RPA-ssDNA complex does not. Finally, we present a model for the early steps of the repair of double-strand DNA breaks in yeast.     

Studies of the interaction between Rad52 protein and the yeast single-stranded DNA binding protein RPA

The RFA1 gene encodes the large subunit of the yeast trimeric single-stranded DNA binding protein replication protein A (RPA), which is known to play a critical role in DNA replication. A Saccharomyces cerevisiae strain carrying the rfa1-44 allele displays a number of impaired recombination and repair phenotypes, all of which are suppressible by overexpression of RAD52. We demonstrate that a rad52 mutation is epistatic to the rfa1-44 mutation, placing RFA1 and RAD52 in the same genetic pathway. Furthermore, two-hybrid analysis indicates the existence of interactions between Rad52 and all three subunits of RPA. The nature of this Rad52-RPA interaction was further explored by using two different mutant alleles of rad52. Both mutations lie in the amino terminus of Rad52, a region previously defined as being responsible for its DNA binding ability (U. H. Mortenson, C. Beudixen, I. Sunjeuaric, and R. Rothstein, Proc. Natl. Acad. Sci. USA 93:10729-10734, 1996). The yeast two-hybrid system was used to monitor the protein-protein interactions of the mutant Rad52 proteins. Both of the mutant proteins are capable of self-interaction but are unable to interact with Rad51. The mutant proteins also lack the ability to interact with the large subunit of RPA, Rfa1. Interestingly, they retain their ability to interact with the medium-sized subunit, Rfa2. Given the location of the mutations in the DNA binding domain of Rad52, a model incorporating the role of DNA in the protein-protein interactions involved in the repair of DNA double-strand breaks is presented.     

Use of asymmetric PCR to generate long primers and single-stranded DNA for incorporating cross-linking analogs into specific sites in a DNA probe

Photoactivatable DNA analogs have been incorporated enzymatically into DNA and used to map the locations of polypeptides in protein complexes bound to DNA. We have developed a procedure for generating long primers from short oligodeoxyribonucleotides (oligos) to incorporate DNA cross-linkers at specific sites within either strand of DNA probes of < or = 206 bp. Single-stranded DNA molecules of 52-206 nucleotides in length were generated by asymmetric polymerase chain reactions (aPCR), using an excess of one short sense-strand primer to be extended and a limiting amount of each short antisense primer that is complementary to and defines the 3' end of the long primer to be generated. The noncross-linking strand of the DNA probe was also generated by aPCR from the DNA sequence of interest. The long primers were annealed to the full-length noncross-linking DNA strand to form a partially double-stranded DNA. Cross-linking analogs and radioactive deoxyribonucleotides (dNTPs), followed by normal dNTPs, were enzymatically incorporated onto the long primers to form the double-stranded DNA cross-linking probes. This method is reproducible and avoids many of the difficulties encountered by other published methods.     

Functional domains of Escherichia coli single-stranded DNA binding protein as assessed by analyses of the deletion mutants

A series of C- and N-terminal deletion mutants of Escherichia coli single-stranded DNA binding protein (SSB) was constructed, purified, and characterized in terms of ability to self-multimerize and to bind to DNA. High-performance gel filtration chromatography revealed that the amino acids 89-105 play a key role in the maintenance of homotetramer for native SSB of 177 amino acids. Interestingly, all of the N-terminal deletion mutants studied here were eluted as octamers, indicating that the N-terminal 11 residues are involved in the prevention of the formation of octamers. The binding of SSB and its deletion mutant proteins to single-stranded d(T)n was examined by gel mobility shift assay and circular dichroism spectroscopy. C-terminal deletion mutant proteins, SSB1-135 and SSB1-115, maintained high affinity and may be wrapped by single-stranded DNA (ssDNA) in the same way as in the case of native SSB. In contrast, deletion of the C-terminal region (residues 89-115) or N-terminal region (residues 1-11) caused a dramatic decrease in the binding affinity. Furthermore, two different stoichiometries of SSB in the complexes with d(T)64, but not with d(T)32, were observed for native SSB, SSB1-135, SSB1-115, and SSB37-177, suggesting that the (SSB)65 and (SSB)35 binding modes, as previously demonstrated [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594-3603; Bujalowski, W., & Lohman, T. M. (1986) Biochemistry 25, 7799-7802], occurred at lower and higher SSB concentrations, respectively. A functional map for SSB molecule was presented and discussed.     

Isolation, sequencing and overproduction of the single-stranded DNA binding protein from Pseudomonas aeruginosa PAO

The effect of gamma-aminobutyric acid (GABA) on intracellular Ca2+ concentration ([Ca2+]i) in cultured prenatal rat cortical neurons was investigated using fluorescence imaging. GABA or muscimol, but not baclofen, increased [Ca2+]i in a dose-dependent manner. The GABAA receptor antagonists, bicuculline and picrotoxin, inhibited the GABA response. Furosemide, an inhibitor of the Na+/K+/2Cl- cotransporter, inhibited the GABA response in a noncompetitive manner. Ethacrynic acid, an inhibitor of an ATP-dependent Cl- pump, also inhibited the GABA-induced increased in [Ca2+]i. These results suggest a role for Cl- transport processes in the GABA response. The coapplication of GABA and high K+ led to a non-additive increase in the GABA response. The GABA response was also inhibited by nifedipine, a voltage-gated Ca2+ channel blocker, and abolished by the absence of extracellular Ca2+. Results indicate that the GABA response shares a common pathway of Ca2+ movement with the high K(+)-induced response. These observations suggest that the stimulation with GABA results in Ca2+ influx through voltage-gated Ca2+ channels, and that these effects are dependent on Cl- transport systems.     

Mutations in the adenovirus-encoded single-stranded DNA binding protein that result in altered accumulation of early and late viral RNAs

Indorenate (TR3369, 5-methoxytryptamine b-methylcarboxylate HCl) is a 5-HT1-like receptor agonist with hypotensive activity. Here, we describe that indorenate also decreases food intake (ED50 26.1 mg/kg) without an appreciable effect in water intake (the estimated ED50 for water was 589.8 mg/kg). The anorectic activity of indorenate was compared to the effects of amphetamine and other serotonin agonists; the effect of indorenate was smaller than those of the other compounds; however, the effect of indorenate was specific to food, whereas all the other drugs also produced significant decrements in water intake. The serotonin antagonists cinanserin, cyproheptadine, methergoline and methysergide effectively prevented the decrease in food intake produced by indorenate and fenfluramine. Haloperidol, a dopaminergic antagonist, was ineffective in preventing the effect of indorenate although it prevented the anorectic effect of amphetamine. The present results suggest the participation of serotoninergic, but not dopaminergic mechanisms, in the decrease in food intake produced by indorenate.     

A phage single-stranded DNA (ssDNA) binding protein complements ssDNA accumulation of a geminivirus and interferes with viral movement

Geminiviruses are plant viruses with circular single-stranded DNA (ssDNA) genomes encapsidated in double icosahedral particles. Tomato leaf curl geminivirus (ToLCV) requires coat protein (CP) for the accumulation of ssDNA in protoplasts and in plants but not for systemic infection and symptom development in plants. In the absence of CP, infected protoplasts accumulate reduced levels of ssDNA and increased amounts of double-stranded DNA (dsDNA), compared to accumulation in the presence of wild-type virus. To determine whether the gene 5 protein (g5p), a ssDNA binding protein from Escherichia coli phage M13, could restore the accumulation of ssDNA, ToLCV that lacked the CP gene was modified to express g5p or g5p fused to the N-terminal 66 amino acids of CP (CP66:6G:g5). The modified viruses led to the accumulation of wild-type levels of ssDNA and high levels of dsDNA. The accumulation of ssDNA was apparently due to stable binding of g5p to viral ssDNA. The high levels of dsDNA accumulation during infections with the modified viruses suggested a direct role for CP in viral DNA replication. ToLCV that produced the CP66:6G:g5 protein did not spread efficiently in Nicotiana benthamiana plants, and inoculated plants developed only very mild symptoms. In infected protoplasts, the CP66:6G:g5 protein was immunolocalized to nuclei. We propose that the fusion protein interferes with the function of the BV1 movement protein and thereby prevents spread of the infection.     

Characterisation of Trypanosoma cruzi populations by DNA polymorphism of the cruzipain gene detected by single-stranded DNA conformation polymorphism (SSCP) and direct sequencing

Fifty fresh isolates of Trypanosoma cruzi from Triatoma dimidiata vectors and 31 from patients with Chagas' disease were analysed for DNA polymorphisms within the 432-bp core region of the cruzipain gene which encodes the active site of cathepsin L-like cystein proteinase. The cruzipain gene showed signs of polymorphism consisting of four different DNA sequences in Central and South American isolates of T. cruzi. The PCR fragments of Guatemalan isolates could be divided into three groups, Groups 1, 2 and 3, based on different patterns of single-stranded DNA conformation polymorphism. All of the strains isolated from Brazil, Chile, and Paraguay, except for the CL strain, showed a Group 4 pattern. Two to four isolates from each group were analysed by cloning and sequencing. A silent mutation occurred between Groups 1 and 2, and five nucleotides and two aa substitutions were detected between Groups 1 and 3. The DNA sequence of Group 4 contained five nucleotides and one aa substitution from Group 1. All of the DNA sequences corresponded well with the single-stranded DNA conformation polymorphism. The Group 1 isolates, the majority in the Guatemalan population (70/81, 86.4%), were isolated from both triatomines and humans, but Group 3 were isolated only from humans. Moreover, the Group 2 isolates were detected only in triatomine vectors (9/50; 18%), but never in humans (0/32, P<0.05) suggesting that this group has an independent life-cycle in sylvatic animals and is maintained by reservoir hosts other than humans.     

Incision at DNA G.T mispairs by extracts of mammalian cells occurs preferentially at cytosine methylation sites and is not targeted by a separate G.T binding reaction

We have investigated the specificities of G.T mismatch binding proteins and of G.T mismatch cleavage in extracts of mammalian cells. G.T mismatch-specific protein:DNA complex formation by cell extracts was independent of the local sequence context of the mismatch. Cell extracts performed similar levels of protein binding to DNA substrates in which a single G.T mispair was preceded by T, G, A, C, or 5-meC. In contrast, incision by extracts of the T-containing strand of a G.T mismatch exhibited a strong sequence specificity and efficient strand cleavage was only observed when the mismatched G was in a CpG sequence. Thus, oligonucleotides containing either CpgGpT or 5meCpGGpT were efficiently incised, but not those containing GpGCpT, ApGTpT, or TpGApT sequences. Cell lines made resistant to the alkylating agent N-methyl-N-nitrosourea have previously been found to be defective in a G.T mismatch binding reaction. The defect in binding by extracts prepared from these cells extended to G.T mismatches in several sequence contexts. The variant extracts nevertheless incised G.T mismatches normally suggesting that this particular binding activity is not required for incision. The data indicate that incision by this activity is targeted to the CpG sequences in which G.T mismatches are formed by the mutagenic deamination of DNA 5-methylcytosine. In this regard the repair pathway resembles the very short patch (vsp) repair pathway in Escherichia coli.     

Termination within oligo(dT) tracts in template DNA by DNA polymerase gamma occurs with formation of a DNA triplex structure and is relieved by mitochondrial single-stranded DNA-binding protein

Xenopus laevis DNA polymerase gamma (pol gamma) exhibits low activity on a poly(dT)-oligo(dA) primer-template. We prepared a single-stranded phagemid template containing a dT41 sequence to test the ability of pol gamma to extend a primer through a defined oligo(dT) tract. pol gamma terminates in the center of this dT41 sequence. This replication arrest is abrogated by addition of single-stranded DNA-binding protein or by substitution of 7-deaza-dATP for dATP. These features are consistent with the formation of a T.A*T DNA triplex involving the primer stem. Replication arrest occurs under conditions that permit highly processive DNA synthesis by pol gamma. A similar replication arrest occurs for T7 DNA polymerase, which is also a highly processive DNA polymerase. These results suggest the possibility that DNA triplex formation can occur prior to dissociation of DNA polymerase. Primers with 3'-oligo(dA) termini annealed to a template with a longer oligo(dT) tract are not efficiently extended by pol gamma unless single-stranded DNA-binding protein is added. Thus, one of the functions of single-stranded DNA-binding protein in mtDNA maintenance may be to enable pol gamma to successfully replicate through dT-rich sequences.     

Competition for DNA binding sites between the short and long forms of E2 dimers underlies repression in bovine papillomavirus type 1 DNA replication control

Papillomaviruses establish a long-term latency in vivo by maintaining their genomes as nuclear plasmids in proliferating cells. Bovine papillomavirus type 1 encodes two proteins required for viral DNA replication: the helicase E1 and the positive regulator E2. The homodimeric E2 is known to cooperatively bind to DNA with E1 to form a preinitiation complex at the origin of DNA replication. The virus also codes for two short forms of E2 that can repress viral functions when overexpressed, and at least one copy of the repressor is required for stable plasmid maintenance in transformed cells. Employing a tetracycline-regulated system to control E1 and E2 production from integrated loci, we show that the short form of E2 negatively regulates DNA replication. We also found that the short form could repress replication in a cell-free replication system and that the repression requires the DNA binding domain of the protein. In contrast, heterodimers of the short and long forms were activators and, by footprint analysis, were shown to be as potent as homodimeric E2 in loading E1 to its cognate site. DNA binding studies show that when E1 levels are low and are dependent upon E2 for occupancy of the origin site, the repressor can block E1-DNA interactions. We conclude that DNA replication modulation results from competition between the different forms of E2 for DNA binding. Given that heterodimers are active and that the repressor form of E2 shows little cooperativity with E1 for DNA binding, this protein is a weak repressor.     

Expression of cell adhesion molecules in placentae from pregnancies complicated by pre-eclampsia and intrauterine growth retardation

Platelets and neutrophils are involved in maternal placental vascular damage in pre-eclampsia. Recruitment of these cells is probably mediated by cell adhesion molecules expressed at the uteroplacental bed. It remains controversial as to whether platelets and neutrophils mediate damage to trophoblast or villous vasculature. The purpose of this study was to determine the expression of cell adhesion molecules in placentae from normal pregnancies and pregnancies complicated by pre-eclampsia and intrauterine growth retardation (IUGR). Immunostaining for platelet endothelial cell adhesion molecule (PECAM) and intercellular adhesion molecule-1 (ICAM-1) was localized mainly to the endothelium of stem villi, intermediate villi, terminal villi and decidual vessels. Scattered staining for ICAM-1 was also evident in the stroma and fetal membranes. The endothelium of stem villi, intermediate villi and terminal villi were all negative for vascular cell adhesion molecule-1 (VCAM-1) and E-Selectin. PECAM, ICAM-1 and ICAM-2 mRNA were all detectable in normal placentae using northern blotting analysis whereas mRNA for E-Selectin and VCAM-1 were both undetectable. There were no differences in cell adhesion molecule immunostaining or mRNA expression in placentae from pregnancies complicated by pre-eclampsia and IUGR inconclusion, expression of cell adhesion molecules in placentae from pre-eclampsia and IUGR are consistent with a normal physiological role in vascular function.     

DNA bending is essential for the site-specific recognition of DNA response elements by the DNA binding domain of the tumor suppressor protein p53

We have used circular permutation assays to determine the extent and location of the DNA bend induced by the DNA binding domain of human wild type p53 (p53DBD) upon binding to several naturally occurring DNA response elements. We have found that p53DBD binding induces axial bending in all of the response elements investigated. In particular, response elements having a d(CATG) sequence at the junction of two consensus pentamers in each half-site favor highly bent complexes (bending angle is approximately 50 degrees ), whereas response elements having d(CTTG) bases at this position are less bent (bending angles from approximately 37 to approximately 25 degrees ). Quantitative electrophoretic mobility shift assays of different complexes show a direct correlation between the DNA bending angle and the binding affinity of the p53DBD with the response elements, i.e. the greater the stability of the complex, the more the DNA is bent by p53DBD binding. The study provides evidence that the energetics of DNA bending, as determined by the presence or absence of flexible sites in the response elements, may contribute significantly to the overall binding affinity of the p53DBD for different sequences. The results therefore suggest that both the structure and the stability of the p53-DNA complex may vary with different response elements. This variability may be correlated with variability in p53 function.     

A common core for binding single-stranded DNA: structural comparison of the single-stranded DNA-binding proteins (SSB) from E. coli and human mitochondria

The crystal structure of the DNA-binding domain of E. coli SSB (EcoSSB) has been determined to a resolution of 2.5 A. This is the first reported structure of a prokaryotic SSB. The structure of the DNA-binding domain of the E. coli protein is compared to that of the human mitochondrial SSB (HsmtSSB). In spite of the relatively low sequence identity between them, the two proteins display a high degree of structural similarity. EcoSSB crystallises with two dimers in the asymmetric unit, unlike HsmtSSB which contains only a dimer. This is probably a consequence of the different polypeptide chain lengths in the EcoSSB heterotetramer. Crucial differences in the dimer-dimer interface of EcoSSB may account for the inability of EcoSSB and HsmtSSB to form cross-species heterotetramers, in contrast to many bacterial SSBs.     

Binding of low mobility group proteins with DNA examined in homologous and heterologous systems by gel retardation assay

In addition to age-related deficits in morphine antinociception in female rats, gender and gonadectomy differences have also been observed, with male rats displaying greater magnitudes of effects than females and castrated males. Since there are little data indicating how aging, gender, and gonadectomy interact in modulating morphine antinociception, the present study evaluated alterations in this response as functions of age (6, 12, 18, and 24 months), gender, and gonadal status (intact, gonadectomized) across a dose range (1-10 mg/kg) and time course (0.5-2 h) on the tail-flick test. The maximal percentage effect (MPE) of morphine (1 mg/kg) was significantly increased in castrated males (18 months), sham females (18 and 24 months), and ovariectomized females (18 months) relative to 6-month-old groups. Increases in the MPE of morphine (1 mg/kg) occurred in sham females (24 months) relative to corresponding sham males and ovariectomized females. The MPE of morphine (2.5 mg/kg) was significantly increased in sham males (18 months) and decreased in sham females (12 months). Decreases in the MPE of morphine (2.5 mg/kg) occurred in castrated males (18 and 24 months) as well as sham (18 months) and ovariectomized (18 and 24 months) females relative to sham males. Whereas the MPE of morphine (5 mg/kg) was unchanged by these variables, the MPE of morphine (10 mg/kg) was significantly decreased in sham females (18 and 24 months) relative to females aged 6 months, as well as males and ovariectomized females aged 24 months.(ABSTRACT TRUNCATED AT 250 WORDS)