Binding of the UvrB dimer to non-damaged and damaged DNA: residues Y92 and Y93 influence the stability of both subunits

UvrB is the ultimate damage-binding protein in bacterial nucleotide excision repair. Previous AFM experiments have indicated that UvrB binds to a damage as a dimer. In this paper we visualize for the first time a UvrB dimer in a gel retardation assay, with the second subunit (B2) more loosely bound than the subunit (B1) that interacts with the damage. A beta-hairpin motif in UvrB plays an important role in damage specific binding. Alanine substitutions of Y92 or Y93 in the beta-hairpin result in proteins that kill E. coli cells as a consequence of incision in non-damaged DNA. Apparently, both residues are needed to prevent binding of UvrB to non-damaged DNA. The lethality of Y93A results from UvrC-mediated incisions, whereas that of Y92A is due to incisions by Cho. This difference could be ascribed to a difference in stability of the B2 subunit in the mutant UvrB-DNA complexes. We show that for 3' incision UvrC needs to displace this second UvrB subunit from the complex, whereas Cho seems capable to incise the dimer-complex. Footprint analysis of the contacts of UvrB with damaged DNA revealed that the B2 subunit interacts with the flanking DNA at the 3' side of the lesion. The B2 subunit of mutant Y92A appeared to be more firmly associated with the DNA, indicating that even when B1 is bound to a lesion, the B2 subunit probes the adjacent DNA for presence of damage. We propose this to be a reflection of the process that the UvrB dimer uses to find lesions in the DNA. In addition to preventing binding to non-damaged DNA, the Y92 and Y93 residues appear also important for making specific contacts (of B1) with the damaged site. We show that the concerted action of the two tyrosines lead to a conformational change in the DNA surrounding the lesion, which is required for the 3' incision reaction.     

Reconstitution of DNA topoisomerase VI of the thermophilic archaeon Sulfolobus shibatae from subunits separately overexpressed in Escherichia coli.

DNA topoisomerase VI from the hyperthermophilic archaeon Sulfolobus shibatae is the prototype of a novel family of type II DNA topoisomerases that share little sequence similarity with other type II enzymes, including bacterial and eukaryal type II DNA topoisomerases and archaeal DNA gyrases. DNA topoisomerase VI relaxes both negatively and positively supercoiled DNA in the presence of ATP and has no DNA supercoiling activity. The native enzyme is a heterotetramer composed of two subunits, A and B, with apparent molecular masses of 47 and 60 kDa, respectively. Here wereport the overexpression in Escherichia coli and the purification of each subunit. The A subunit exhibits clusters of arginines encoded by rare codons in E.coli . The expression of this protein thus requires the co-expression of the minor E.coli arginyl tRNA which reads AGG and AGA codons. The A subunit expressed in E.coli was obtained from inclusion bodies after denaturation and renaturation. The B subunit was overexpressed in E.coli and purified in soluble form. When purified B subunit was added to the renatured A subunit, ATP-dependent relaxation and decatenation activities of the hyperthermophilic DNA topoisomerase were reconstituted. The reconstituted recombinant enzyme exhibits a specific activity similar to the enzyme purified from S.shibatae . It catalyzes transient double-strand cleavage of DNA and becomes covalently attached to the ends of the cleaved DNA. This cleavage is detected only in the presence of both subunits and in the presence of ATP or its non-hydrolyzable analog AMPPNP.     

Subunit composition of DNA polymerases A and B from wheat cell

DNA polymerases A and B purified from wheat (Triticum monococcum) embryos were previously shown to be respectively the plant counterparts of mammalian DNA polymerases α and δ. From wheat cultured cells, we isolated a protein fraction able to replicate a DNA template/primer in a cell-free DNA replication assay. This fraction contains the DNA polymerases pol A and pol B, exhibiting the same biochemical properties as those found in wheat embryo. The catalytic subunits of DNA polymerases pol A and B purified from this fraction were analysed by a DNA polymerase trap assay and their molecular mass were respectively determined as 90 and 125 kDa. This shows that pol A catalytic subunit is shorter than those of yeast or mammal DNA polymerases α (respectively 180 and 165 kDa), whereas pol B catalytic subunit exhibits the same molecular mass as yeast and mammal DNA polymerases δ (125 kDa). Catalytic subunit identification using DNA polymerase trap assay could be a good alternative to isolate and sequence active polypeptides from low purified enzymes. These results contribute to the molecular characterization of DNA replication enzymes in plants and will permit to establish a plant DNA replication model.     

The role of the 70 kDa subunit of human DNA polymerase alpha in DNA replication.

DNA polymerase alpha is the only enzyme in eukaryotic cells capable of starting DNA chains de novo and is required for the initiation of SV40 DNA replication in vitro. We have cloned the 70 kDa subunit of human DNA polymerase alpha (hereafter referred to as the B subunit) and expressed it as a fusion protein in bacteria. The purified fusion protein forms a stable complex with SV40 T antigen, both in solution and when T antigen is bound to the SV40 origin of DNA replication. Analysis of mutant forms of the B subunit indicates that the N-terminal 240 amino acids are sufficient to mediate complex formation. The B subunit fusion protein promotes formation of a complex containing T antigen and the catalytic subunit (subunit A) of DNA polymerase alpha, suggesting that it serves to tether the two proteins. These physical interactions are functionally significant, since the ability of T antigen to stimulate the activity of the catalytic subunit of DNA polymerase alpha is highly dependent upon the B subunit. We suggest that the interactions mediated by the B subunit play an important role in SV40 DNA replication by promoting DNA chain initiation at the origin and/or facilitating the subsequent priming and synthesis of DNA chains on the lagging strand template. The protein may play similar roles in cellular DNA replication.     

Different oligomeric forms of protein phosphatase 2A activate and inhibit simian virus 40 DNA replication.

The ability of simian virus 40 (SV40) large T antigen to catalyze the initiation of viral DNA replication is regulated by its phosphorylation state. Previous studies have identified the free catalytic subunit of protein phosphatase 2A (PP2Ac) as the cellular phosphatase which can remove inhibitory phosphoryl groups from serines 120 and 123. The catalytic C subunit exists in the cell complexed with a 65-kDa A subunit and one of several B subunits. To determine if any of the holoenzymes could activate T antigen, we tested the ability of the heterodimeric AC and two heterotrimeric ABC forms to stimulate T-antigen function in unwinding the origin of SV40 DNA replication. Only free catalytic subunit C and the heterotrimeric form with a 72-kDa B subunit (PP2A-T72) could stimulate T-antigen-dependent origin unwinding. Both the dimeric form (PP2A-D) and the heterotrimer with a 55-kDa B subunit (PP2A-T55) actively inhibited T-antigen function. We found that PP2A-T72 activated T antigen by dephosphorylating serines 120 and 123, while PP2A-D and PP2A-T55 inactivated T antigen by dephosphorylating the p34cdc2 target site, threonine 124. Thus, alterations in the subunit composition of PP2A holoenzymes have significant functional consequences for the initiation of in vitro SV40 DNA replication. The regulatory B subunits of PP2A may play a role in regulating SV40 DNA replication in infected cells as well.     

Antagonism of the B subunit of DNA gyrase eliminates plasmids pBR322 and pMG110 from Escherichia coli.

The constructed plasmid pBR322 and the native plasmid pMG110 were eliminated (cured) from growing Escherichia coli cells by the antagonism of the B subunit of the bacterial enzyme DNA gyrase. The antagonism may be by the growth of cells (i) at semipermissive temperatures in a bacterial mutant containing a thermolabile gyrase B subunit or (ii) at semipermissive concentrations of coumermycin A1, an antibiotic that specifically inhibits the B subunit of DNA gyrase. The kinetics of plasmid elimination indicate that plasmid loss occurs too rapidly to be explained solely by the faster growth of that plasmid-free bacteria and, therefore, represents interference with plasmid maintenance.     

Tryptic fragments of the Escherichia coli DNA gyrase A protein

Treatment of the Escherichia coli DNA gyrase A protein with trypsin generates two large fragments which are stable to further digestion. The molecular masses of these fragments are 64 and 33 kDa, and they are shown to be derived from the N terminus and the C terminus of the A protein, respectively. These fragments could represent structural and/or functional domains within the A subunit of DNA gyrase. The trypsin-cleaved A protein (A'), in combination with the B subunit of gyrase, can support ATP-dependent supercoiling of relaxed DNA and other reactions of DNA gyrase. The isolated 64-kDa fragment will also catalyse DNA supercoiling in the presence of the B protein, but the 33-kDa fragment shows no enzymic activities. We conclude that the N-terminal 64-kDa fragment represents the DNA breakage/reunion domain of the A protein, while the 33-kDa fragment may contribute to the stability of the gyrase-DNA complex.     

Mitogenesis of 3T3 fibroblasts induced by endogenous ganglioside is not mediated by cAMP, protein kinase C, or phosphoinositides turnover

The B subunit of cholera toxin, which binds specifically to ganglioside GM1, stimulates DNA synthesis in quiescent Swiss 3T3 fibroblasts grown in chemically defined medium. The mitogenic response to the B subunit was potentiated by insulin and other growth factors. To elucidate the mechanism by which the B subunit stimulates cell growth , its effects on several transmembrane signaling systems which have been suggested to play a vital role in cell growth regulation were examined. The B subunit did not increase cAMP levels nor activate adenylate cyclase. The B subunit induced a rapid and profound increase in intracellular free Ca2+ as measured with the fluorescent Ca2+-sensitive dye quin 2/AM. Removal of external Ca2+ completely inhibited the signal, thus suggesting that the B subunit elevates intracellular Ca2+ through a net influx of extracellular Ca2+ rather than by causing the release of Ca2+ from intracellular stores. These findings are consistent with the observations that the B subunit induced reinitiation of DNA synthesis without activation of phospholipase C. There was no increase in the formation of inositol trisphosphate, the second messenger that mediates release of Ca2+ from intracellular stores. In addition, the B subunit still stimulated DNA synthesis in Swiss 3T3 cells pretreated with phorbol ester to down-regulate protein kinase C. These results suggest that the mitogenic effects of the B subunit are mediated mainly by facilitation of Ca2+ influx and that activations of adenylate cyclase, phospholipase C, or protein kinase C are not obligatory steps in the initiation of cell growth by the B subunit. Furthermore, the observation that Ca2+ ionophores, such as ionomycin and A23187, are not mitogenic implies that additional undefined growth signaling pathways may exist in this system.     

Regulation of proliferation by the cholera toxin B subunit in FRTL-5 cells may involve a mechanism independent from the modulation of membrane receptor function

In quiescent rat thyroid (FRTL-5) cells, the B subunit of cholera toxin, which binds to cell surface ganglioside GM1 specifically, alone induced DNA synthesis and markedly enhanced that induced by insulin in serum-free medium. On the other hand, the B subunit inhibited DNA synthesis induced by thyrotropin (TSH). The B subunit did not activate adenylate cyclase and had no effect on the TSH-induced cyclic adenosine 3',5'-monophosphate (cAMP) production. Moreover, the B subunit inhibited DNA synthesis induced by dibutyryl cAMP (Bt2cAMP) or phorbol-12-myristate-13-acetate (PMA). These data demonstrate that the B subunit has both stimulatory and inhibitory effects on DNA synthesis in FRTL-5 cells depending on the presence of other growth factors and that these effects on cell proliferation by the interaction of the B subunit, possibly with cell surface ganglioside GM1, may involve a mechanism independent from the modulation of membrane receptor function through interaction with growth factor receptor.     

The C-terminal domain of the Escherichia coli DNA gyrase A subunit is a DNA-binding protein.

We have constructed a clone which over-produces a 33 kDa protein representing the C-terminal portion of the Escherichia coli DNA gyrase A subunit. This protein has no enzymic activity of its own, but will form a complex with a 64 kDa protein (representing the N-terminal part of the A subunit) and the gyrase B subunit, that will efficiently catalyse DNA supercoiling. We show that the 33 kDa protein can bind to DNA on its own in a manner which induces positive supercoiling of the DNA. We propose that the 33 kDa protein represents a domain of the gyrase A subunit which is involved in the wrapping of DNA around DNA gyrase.     

Molecular cloning of a cDNA encoding mouse DNA helicase B, which has homology to Escherichia coli RecD protein, and identification of a mutation in the DNA helicase B from tsFT848 temperature-sensitive DNA replication mutant cells

DNA helicase B is a major DNA helicase in mouse FM3A cells. A temperature-sensitive mutant defective in DNA replication, tsFT848, isolated from FM3A cells, has a heat-labile DNA helicase B. In this study, we purified DNA helicase B from mouse FM3A cells and determined partial amino acid sequences of the purified protein. By using a DNA probe synthesized according to one of the partial amino acid sequences, a cDNA was isolated, which encoded a 121.5 kDa protein containing seven conserved motifs for DNA/RNA helicase superfamily members. A database search revealed similarity between DNA helicase B and the α subunit of exodeoxyribonuclease V of a number of prokaryotes including Escherichia coli RecD protein, but no homologous protein was found in yeast. The cDNA encoding DNA helicase B from tsFT848 was sequenced and a mutation was found between DNA/RNA helicase motifs IV and V.     

Cloning and structural analysis of DNA encoding an A2B1a subunit of glycinin

The partial DNA sequence of a glycinin gene in a genomic clone and a homologous cDNA clone were determined. They have nearly identical nucleotide sequences and encode the basic polypeptide and part of the acidic polypeptide for an A2B1a glycinin subunit. The protein primary structure deduced from the DNA sequence is in close agreement with the amino acid sequence of the subunit determined chemically and confirms assignment of part of the amino acid sequence in the basic component where we were able to establish an overlap using conventional approaches. The coding part of the basic subunit is interrupted by a 625-base pair A + T-rich intron whose boundaries correlate with the established consensus sequences for the exon-intron junctions. Comparison of the nucleotide sequence of the basic subunit of pea legumin gene with that of the gene for A2B1a subunit reveals 70% homology in coding regions, although there is considerably less in the 3'-flanking regions.     

Involvement of host DNA gyrase in growth of bacteriophage T5.

Bacteriophage T5 did not grow at the nonpermissive temperature of 42 degrees C in Escherichia coli carrying a temperature-sensitive mutation in gyrB [gyrB(Ts)], but it did grow in gyrA(Ts) mutants at 42 degrees C. These findings indicate that the A subunit of host DNA gyrase is unnecessary, whereas the B subunit is necessary for growth of T5. The necessity for the B subunit was confirmed by a strong inhibition of T5 growth by novobiocin and coumermycin A1, which interfere specifically with the function of the B subunit of host DNA gyrase. However, T5 growth was also strongly inhibited by nalidixic acid, which interferes specifically with the function of the A subunit. This inhibition was due to the interaction of nalidixic acid with the A subunit and not just to its binding to DNA, because appropriate mutations in the gyrA gene of the host conferred nalidixic acid resistance to the host and resistance to T5 growth in such a host. The inhibition by nalidixic acid was also not due to a cell poison formed between nalidixic acid and the A subunit (K. N. Kreuzer and N. R. Cozzarelli, J. Bacteriol. 140:424-435, 1979) because nalidixic acid inhibited growth of T5 in a gyrA(Ts) mutant (KNK453) at 42 degrees C. We suggest that T5 grows in KNK453 at 42 degrees C because its gyrA(Ts) mutation is leaky for T5. Inhibition of T5 growth due to inactivation of host DNA gyrase was caused mainly by inhibition of T5 DNA replication. In addition, however, late T5 genes were barely expressed when host DNA gyrase was inactivated.     

Structure and function of the (A)BC excinuclease of Escherichia coli

(A)BC excinuclease is the enzymatic activity resulting from the mixture of E. coli UvrA, UvrB and UvrC proteins with damaged DNA. This is a functional definition as new evidence suggests that the three proteins never associate in a ternary complex. The UvrA subunit associates with the UvrB subunit in the form of an A2B1 complex which, guided by UvrA's affinity for damaged DNA binds to a lesion in DNA and delivers the UvrB subunit to the damaged site. The UvrB-damaged DNA complex is extremely stable (t1/2 congruent to 100 min). The UvrC subunit, which has no specific affinity for damaged DNA, recognizes the UvrB-DNA complex with high specificity and the protein complex consisting of UvrB and UvrC proteins makes two incisions, the 8th phosphodiester bond 5' and the 5th phosphodiester bond 3' to the damaged nucleotide. (A)BC excinuclease recognizes DNA damage ranging from AP sites and thymine glycols to pyrimidine dimers, and the adducts of psoralen, cisplatinum, mitomycin C, 4-nitroquinoline oxide and interstrand crosslinks.     

Analysis of yeast RNA polymerases with subunit-specific antibodies

Specific antibodies directed against each polypeptide component of yeast RNA polymerases A or B were prepared and their affinity spectrum determined by protein blot immunodetection. The majority of enzyme A or B subunits were specifically recognized by their respective antiserum. A direct correspondence was established between the polypeptides immunologically related in the three forms of RNA polymerases A, B, and C by reacting the different antibodies with enzymes subunits transferred to a nitrocellulose membrane. Subunit-specific antibodies and antibodies to native enzymes A and B were used to probe the activity of RNA polymerases A, B, and C. Based on DNA protection experiments, the largest subunit of enzymes A and B as well as the common subunit ABC23 appear to be involved in DNA binding.     

Changed properties of the A subunit in DNA gyrase with a B subunit mutation

Summary To investigate the interaction of subunits A and B of DNA gyrase during DNA supercoiling, a Cou^r mutant of Escherichia coli was obtained and the effect of nalidixic acid on the supercoiling of DNA by wild-type and mutant enzymes was assayed. The enzyme of the Cou^r strain proved to be more sensitive to nalidixic acid than the wild-type DNA gyrase. Hence the mutation affecting the B subunit can also change the properties of the A subunit, which fact suggests that the two subunits of DNA gyrase are in contact during DNA supercoiling.     

Molecular model of the A subunit of protein phosphatase 2A: interaction with other subunits and tumor antigens.

Protein phosphatase 2A consists of three subunits, the catalytic subunit (C) and two regulatory subunits (A and B). The A subunit has a rod-like shape and consists of 15 nonidentical repeats. It binds the catalytic subunit through repeats 11 to 15 at the C terminus and the tumor antigens encoded by small DNA tumor viruses through overlapping but distinct regions at N-terminal repeats 2 to 8. A model of the A subunit was developed on the basis of the fact that uncharged or hydrophobic amino acids are conserved at eight defined positions within each repeat. Helical wheel projections suggested that each repeat can be arranged as two interacting amphipathic helixes connected by a short loop. Mutational analysis of the A subunit revealed that the proposed loops are important for binding of tumor antigens, the B subunit, and the C subunit. Native gel analysis of mutant A subunits synthesized in vitro demonstrated that the binding region for the B subunit, previously thought to include repeats 2 to 8, covers repeats 1 to 10 and that the B and C subunits cooperate in binding to the A subunit.     

Cloning and structure of the gene for the subunits of aspartokinase II from Bacillus subtilis

A library of Bacillus subtilis DNA in lambda Charon 4A (Ferrari, E., Henner, D.J., and Hoch, J.A. (1981) J. Bacteriol. 146, 430-432) was screened by an immunological procedure for DNA sequences encoding aspartokinase II of B. subtilis, an enzyme composed of two nonidentical subunits arranged in an alpha 2 beta 2 structure (Moir, D., and Paulus, H. (1977a) J. Biol. Chem. 252, 4648-4654). A recombinant bacteriophage was identified that harbored an 18-kilobase B. subtilis DNA fragment containing the coding sequences for both aspartokinase subunits. The coding sequence for aspartokinase II was subcloned into bacterial plasmids. In response to transformation with the recombinant plasmids, Escherichia coli produced two polypeptides immunologically related to B. subtilis aspartokinase II with molecular weights (43,000 and 17,000) indistinguishable from those found in enzyme produced in B. subtilis. Peptide mapping by partial proteolysis confirmed the identity of the polypeptides produced by the transformed E. coli cells with the B. subtilis aspartokinase II subunits. The size of the cloned B. subtilis DNA fragment could be reduced to 2.9 kilobases by cleavage with PstI restriction endonuclease without affecting its ability to direct the synthesis of complete aspartokinase II subunits, irrespective of its orientation in the plasmid vector. Further subdivision by cleavage with BamHI restriction endonuclease resulted in the production of truncated aspartokinase subunits, each shortened by the same extent. This suggested that a single DNA sequence encoded both aspartokinase subunits and provided an explanation for the earlier observation that the smaller beta subunit of aspartokinase II was highly homologous or identical with the carboxyl-terminal portion of the alpha subunit (Moir, D., and Paulus, H. (1977b) J. Biol. Chem. 252, 4655-4661). A map of the gene for B. subtilis aspartokinase II is proposed in which the coding sequence for the smaller beta subunit overlaps in the same reading frame the promoter-distal portion of the coding sequence for the alpha subunit.     

The accessory subunit B of DNA polymerase gamma is required for mitochondrial replisome function

The mitochondrial replication machinery in human cells includes the DNA polymerase γ holoenzyme and the TWINKLE helicase. Together, these two factors form a processive replication machinery, a replisome, which can use duplex DNA as template to synthesize long stretches of single-stranded DNA. We here address the importance of the smaller, accessory B subunit of DNA polymerase γ and demonstrate that this subunit is absolutely required for replisome function. The duplex DNA binding activity of the B subunit is needed for coordination of POLγ holoenzyme and TWINKLE helicase activities at the mtDNA replication fork. In the absence of proof for direct physical interactions between the components of the mitochondrial replisome, these functional interactions may explain the strict interdependence of TWINKLE and DNA polymerase γ for mitochondrial DNA synthesis. Furthermore, mutations in TWINKLE as well as in the catalytic A and accessory B subunits of the POLγ holoenzyme, may cause autosomal dominant progressive external ophthalmoplegia, a disorder associated with deletions in mitochondrial DNA. The crucial importance of the B subunit for replisome function may help to explain why mutations in these three proteins cause an identical syndrome.