Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2.5-A resolution: structural basis for thermostability.

The crystal structure of the large fragment of the Thermus aquaticus DNA polymerase (Klentaq1), determined at 2.5-A resolution, demonstrates a compact two-domain architecture. The C-terminal domain is identical in fold to the equivalent region of the Klenow fragment of Escherichia coli DNA polymerase I (Klenow pol I). Although the N-terminal domain of Klentaq1 differs greatly in sequence from its counterpart in Klenow pol I, it has clearly evolved from a common ancestor. The structure of Klentaq1 reveals the strategy utilized by this protein to maintain activity at high temperatures and provides the structural basis for future improvements of the enzyme.

RNA polymerase II/III transcription specificity determined by TATA box orientation.

The TATA box sequence in eukaryotes is located about 25 bp upstream of many genes transcribed by RNA polymerase II (Pol II) and some genes transcribed by RNA polymerase III (Pol III). The TATA box is recognized in a sequence-specific manner by the TATA box-binding protein (TBP), an essential factor involved in the initiation of transcription by all three eukaryotic RNA polymerases. We have investigated the recognition of the TATA box by the Pol II and Pol III basal transcription machinery and its role in establishing the RNA polymerase specificity of the promoter. Artificial templates were constructed that contained a canonical TATA box as the sole promoter element but differed in the orientation of the 8-bp TATA box sequence. As expected, Pol II initiated transcription in unfractionated nuclear extracts downstream of the "forward" TATA box. In distinct contrast, transcription that initiated downstream of the "reverse" TATA box was carried out specifically by Pol III. Importantly, this effect was observed regardless of the source of the DNA either upstream or downstream of the TATA sequence. These findings suggest that TBP may bind in opposite orientations on Pol II and Pol III promoters and that opposite, yet homologous, surfaces of TBP may be utilized by the Pol II and Pol III basal machinery for the initiation of transcription.

Interaction of herpes simplex virus 1 origin-binding protein with DNA polymerase alpha.

The herpes simplex virus 1 (HSV-1) genome encodes seven polypeptides that are required for its replication. These include a heterodimeric DNA polymerase, a single-strand-DNA-binding protein, a heterotrimeric helicase/primase, and a protein (UL9 protein) that binds specifically to an HSV-1 origin of replication (oris). We demonstrate here that UL9 protein interacts specifically with the 180-kDa catalytic subunit of the cellular DNA polymerase alpha-primase. This interaction can be detected by immunoprecipitation with antibodies directed against either of these proteins, by gel mobility shift of an oris-UL9 protein complex, and by stimulation of DNA polymerase activity by the UL9 protein. These findings suggest that enzymes required for cellular DNA replication also participate in HSV-1 DNA replication.

Proofreading-defective DNA polymerase II increases adaptive mutation in Escherichia coli.

The role of Escherichia coli DNA polymerase (Pol) II in producing or avoiding mutations was investigated by replacing the chromosomal Pol II gene (polB+) by a gene encoding an exonuclease-deficient mutant Pol II (polBex1). The polBex1 allele increased adaptive mutations on an episome in nondividing cells under lactose selection. The presence of a Pol III antimutator allele (dnaE915) reduced adaptive mutations in both polB+ cells and cells deleted for polB (polB delta 1) to below the wild-type level, suggesting that both Pol II and Pol III are synthesizing episomal DNA in nondividing cells but that in wild-type cells Pol III generates the adaptive mutations. The adaptive mutations were mainly -1 frame-shifts occurring in short homopolymeric runs and were similar in wild-type, polB delta 1, and polBex1 strains. Mutations produced by both Pol III and Pol II ex1 were corrected by the mutHLS mismatch repair system.

Specific binding of RNA polymerase II to the human immunodeficiency virus trans-activating region RNA is regulated by cellular cofactors and Tat.

The regulation of human immunodeficiency virus type 1 (HIV-1) gene expression in response to Tat is dependent on an element downstream of the HIV-1 transcriptional initiation site designated the trans-activating region (TAR). TAR forms a stable stem-loop RNA structure in which a 3-nt bulge structure and a 6-nt loop structure are important for Tat activation. In the absence of Tat, the HIV-1 promoter generates so-called short or nonprocessive transcripts terminating at +60, while in the presence of Tat the synthesis of these short transcripts is markedly decreased and transcripts that extend through the 9.0-kb HIV-1 genome are synthesized. Tat effects on transcriptional elongation are likely due to alterations in the elongation properties of RNA polymerase II. In this study we demonstrated that a set of cellular cofactors that modulate the binding of the cellular protein TRP-185 to the TAR RNA loop sequences also functioned to markedly stimulate the specific binding of hypophosphorylated (IIa) and hyperphosphorylated (IIo) RNA polymerase II to TAR RNA. The concentrations of RNA polymerase II required for this interaction with TAR RNA were similar to those required to initiate in vitro transcription from the HIV-1 long terminal repeat. RNA gel retardation analysis with wild-type and mutant TAR RNAs indicated that the TAR RNA loop and bulge sequences were critical for the binding of RNA polymerase II. The addition of wild-type but not mutant Tat protein to gel retardation analysis with TAR RNA and RNA polymerase II resulted in the loss of binding of RNA polymerase II binding to TAR RNA. These results suggest that Tat may function to alter RNA polymerase II, which is paused due to its binding to HIV-1 TAR RNA with resultant stimulation of its transcriptional elongation properties.

The choice of alternative 5' splice sites in influenza virus M1 mRNA is regulated by the viral polymerase complex.

The influenza virus M1 mRNA has two alternative 5' splice sites: a distal 5' splice site producing mRNA3 that has the coding potential for 9 amino acids and a proximal 5' splice site producing M2 mRNA encoding the essential M2 ion-channel protein. Only mRNA3 was made in uninfected cells transfected with DNA expressing M1 mRNA. Similarly, using nuclear extracts from uninfected cells, in vitro splicing of M1 mRNA yielded only mRNA3. Only when the mRNA3 5' splice site was inactivated by mutation was M2 mRNA made in uninfected cells and in uninfected cell extracts. In influenza virus-infected cells, M2 mRNA was made, but only after a delay, suggesting that newly synthesized viral gene product(s) were needed to activate the M2 5' splice site. We present strong evidence that these gene products are the complex of the three polymerase proteins, the same complex that functions in the transcription and replication of the viral genome. Gel shift experiments showed that the viral polymerase complex bound to the 5' end of the viral M1 mRNA in a sequence-specific and cap-dependent manner. During in vitro splicing catalyzed by uninfected cell extracts, the binding of the viral polymerase complex blocked the mRNA3 5' splice site, resulting in the switch to the M2 mRNA 5' splice site and the production of M2 mRNA.

A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides.

Bacteriophage T7 DNA polymerase efficiently incorporates a chain-terminating dideoxynucleotide into DNA, in contrast to the DNA polymerases from Escherichia coli and Thermus aquaticus. The molecular basis for this difference has been determined by constructing active site hybrids of these polymerases. A single hydroxyl group on the polypeptide chain is critical for selectivity. Replacing tyrosine-526 of T7 DNA polymerase with phenylalanine increases discrimination against the four dideoxynucleotides by > 2000-fold, while replacing the phenylalanine at the homologous position in E. coli DNA polymerase I (position 762) or T. aquaticus DNA polymerase (position 667) with tyrosine decreases discrimination against the four dideoxynucleotides 250- to 8000-fold. These mutations allow the engineering of new DNA polymerases with enhanced properties for use in DNA sequence analysis.

DNA polymerase beta bypasses in vitro a single d(GpG)-cisplatin adduct placed on codon 13 of the HRAS gene.

We have examined the capacity of calf thymus DNA polymerases alpha, beta, delta, and epsilon to perform in vitro translesion synthesis on a substrate containing a single d(GpG)-cisplatin adduct placed on codon 13 of the human HRAS gene. We found that DNA synthesis catalyzed by DNA polymerases alpha, delta, and epsilon was blocked at the base preceding the lesion. Addition of proliferating cell nuclear antigen to DNA polymerase delta and replication protein A to DNA polymerase alpha did not restore their capacity to elongate past the adduct. On the other hand, DNA polymerase beta efficiently bypassed the cisplatin adduct. Furthermore, we observed that DNA polymerase beta was the only polymerase capable of primer extension of a 3'-OH located opposite the base preceding the lesion. Likewise, DNA polymerase beta was able to elongate the arrested replication products of the other three DNA polymerases, thus showing its capacity to successfully compete with polymerases alpha, delta, and epsilon in the stalled replication complex. Our data suggest (i) a possible mechanism enabling DNA polymerase beta to bypass a d(GpG)-cisplatin adduct in vitro and (ii) a role for this enzyme in processing DNA damage in vivo.

A dominant-negative mutant of human poly(ADP-ribose) polymerase affects cell recovery, apoptosis, and sister chromatid exchange following DNA damage.

Poly(ADP-ribose) polymerase [PARP; NAD+ ADP-ribosyltransferase; NAD+:poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] is a zinc-dependent eukaryotic DNA-binding protein that specifically recognizes DNA strand breaks produced by various genotoxic agents. To study the biological function of this enzyme, we have established stable HeLa cell lines that constitutively produce the 46-kDa DNA-binding domain of human PARP (PARP-DBD), leading to the trans-dominant inhibition of resident PARP activity. As a control, a cell line was constructed, producing a point-mutated version of the DBD, which has no affinity for DNA in vitro. Expression of the PARP-DBD had only a slight effect on undamaged cells but had drastic consequences for cells treated with genotoxic agents. Exposure of cell lines expressing the wild-type (wt) or the mutated PARP-DBD, with low doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in an increase in their doubling time, a G2 + M accumulation, and a marked reduction in cell survival. However, UVC irradiation had no preferential effect on the cell growth or viability of cell lines expressing the PARP-DBD. These PARP-DBD-expressing cells treated with MNNG presented the characteristic nucleosomal DNA ladder, one of the hallmarks of cell death by apoptosis. Moreover, these cells exhibited chromosomal instability as demonstrated by higher frequencies of both spontaneous and MNNG-induced sister chromatid exchanges. Surprisingly, the line producing the mutated DBD had the same behavior as those producing the wt DBD, indicating that the mechanism of action of the dominant-negative mutant involves more than its DNA-binding function. Altogether, these results strongly suggest that PARP is an element of the G2 checkpoint in mammalian cells.

Formation of brome mosaic virus RNA-dependent RNA polymerase in yeast requires coexpression of viral proteins and viral RNA.

In this report we show that yeast expressing brome mosaic virus (BMV) replication proteins 1a and 2a and replicating a BMV RNA3 derivative can be extracted to yield a template-dependent BMV RNA-dependent RNA polymerase (RdRp) able to synthesize (-)-strand RNA from BMV (+)-strand RNA templates added in vitro. This virus-specific yeast-derived RdRp mirrored the template selectivity and other characteristics of RdRp from BMV-infected plants. Equivalent extracts from yeast expressing 1a and 2a but lacking RNA3 contained normal amounts of 1a and 2a but had no RdRp activity on BMV RNAs added in vitro. To determine which RNA3 sequences were required in vivo to yield RdRp activity, we tested deletions throughout RNA3, including the 5',3', and intercistronic noncoding regions, which contain the cis-acting elements required for RNA3 replication in vivo. RdRp activity was obtained only from cells expressing 1a, 2a, and RNA3 derivatives retaining both 3' and intercistronic noncoding sequences. Strong correlation between extracted RdRp activity and BMV (-)-strand RNA accumulation in vivo was found for all RNA3 derivatives tested. Thus, extractable in vitro RdRp activity paralleled formation of a complex capable of viral RNA synthesis in vivo. The results suggest that assembly of active RdRp requires not only viral proteins but also viral RNA, either to directly contribute some nontemplate function or to recruit essential host factors into the RdRp complex and that sequences at both the 3'-terminal initiation site and distant internal sites of RNA3 templates may participate in RdRp assembly and initiation of (-)-strand synthesis.

Rapid RNA polymerase genetics: one-day, no-column preparation of reconstituted recombinant Escherichia coli RNA polymerase.

We present a simple, rapid procedure for reconstitution of Escherichia coli RNA polymerase holoenzyme (RNAP) from individual recombinant alpha, beta, beta', and sigma 70 subunits. Hexahistidine-tagged recombinant alpha subunit purified by batch-mode metal-ion-affinity chromatography is incubated with crude recombinant beta, beta', and sigma 70 subunits from inclusion bodies, and the resulting reconstituted recombinant RNAP is purified by batch-mode metal-ion-affinity chromatography. RNAP prepared by this procedure is indistinguishable from RNAP prepared by conventional methods with respect to subunit stoichiometry, alpha-DNA interaction, catabolite gene activator protein (CAP)-independent transcription, and CAP-dependent transcription. Experiments with alpha (1-235), an alpha subunit C-terminal deletion mutant, establish that the procedure is suitable for biochemical screening of subunit lethal mutants.

NusA interferes with interactions between the nascent RNA and the C-terminal domain of the alpha subunit of RNA polymerase in Escherichia coli transcription complexes.

The effects of NusA on the RNA polymerase contacts made by nucleotides at internal positions in the nascent RNA in Escherichia coli transcription complexes were analyzed by using the photocrosslinking nucleotide analog 5-[(4-azidophenacyl) thio]-UMP. It was placed at nucleotides between +6 and +15 in RNA transcribed from the phage lambda PR' promoter. Crosslinks of analog in these positions in RNAs which contained either 15, 28, 29, or 49 nt were examined. Contacts between the nascent RNA and proteins in the transcription complex were analyzed as the RNA was elongated, by placing the crosslinker nearest the 5' end of the RNA 10, 23, 24, or 44 nt away from the 3' end. The beta or beta' subunit of polymerase, and NusA when added, were contacted by RNA from 15 to 49 nt long. When the upstream crosslinker was 24 nt from the 3" end of the RNA (29-nt RNA), alpha was also contacted in the absence of NusA. The addition of NusA prevented RNA crosslinking to alpha. When the crosslinker was 44 nt from the 3' end (49-nt RNA), alpha crosslinks were still observed, but crosslinks to beta or beta' and NusA were greatly diminished. RNA crosslinking to alpha, and loss of this crosslink when NusA was added, was observed in the presence of NusB, NusE, and NusG and when transcription was carried out in the presence of an E. coli S100 cell extract. Peptide mapping localized the RNA interactions to the C-terminal domain of alpha.

General requirement for RNA polymerase II holoenzymes in vivo.

Yeast RNA polymerase II holoenzymes have been described that consist of RNA polymerase II, a subset of general transcription factors, and nine SRB regulatory proteins. The feature that distinguishes the RNA polymerase II holoenzymes from other forms of RNA polymerase II in the cell is their tight association with SRB proteins. We investigated the fraction of genes that require SRB proteins in vivo by examining the effect of temperature-sensitive mutations in SRB genes on transcription by RNA polymerase II. Upon transfer to the restrictive temperature, there is a rapid and general shutdown of mRNA synthesis in srb mutant cells. These data, combined with the observation that essentially all of the SRB protein in cells is tightly associated with RNA polymerase II molecules, argue that SRB-containing holoenzymes are the form of RNA polymerase II recruited to most promoters in the cell.

Assembly of functional Escherichia coli RNA polymerase containing beta subunit fragments.

The Escherichia coli rpoB gene, which codes for the 1342-residue beta subunit of RNA polymerase (RNAP), contains two dispensable regions centered around codons 300 and 1000. To test whether these regions demarcate domains of the RNAP beta subunit, fragments encoded by segments of rpoB flanking the dispensable regions were individually overexpressed and purified. We show that these beta-subunit polypeptide fragments, when added with purified recombinant beta', sigma, and alpha subunits of RNAP, reconstitute a functional enzyme in vitro. These results demonstrate that the beta subunit is composed of at least three distinct domains and open another avenue for in vitro studies of RNAP assembly and structure.

Intrinsic transcript cleavage activity of RNA polymerase.

The GreA and GreB transcript cleavage factors of Escherichia coli suppress elongation arrest and may have a proofreading role in transcription. With the use of E. coli greA-greB- mutant, RNA polymerase is demonstrated to possess substantial intrinsic transcript cleavage activity. Mildly alkaline pH mimics the effect of the Gre proteins by inducing transcript cleavage in ternary complexes and antagonizing elongation arrest through a cleavage-and-restart reaction. Thus, transcript cleavage constitutes the second enzymological activity of RNA polymerase along with polymerization/pyrophosphorolysis of RNA, whereas the Gre proteins merely enhance this intrinsic property.