DNA Polymerase β Ribonucleotide Discrimination: INSERTION,MISINSERTION, EXTENSION,AND CODING*

DNA polymerases must select nucleotides that preserve Watson-Crick base pairing rules and choose substrates with the correct (deoxyribose) sugar. Sugar discrimination represents a great challenge because ribonucleotide triphosphates are present at much higher cellular concentrations than their deoxy-counterparts. Although DNA polymerases discriminate against ribonucleotides, many therapeutic nucleotide analogs that target polymerases have sugar modifications, and their efficacy depends on their ability to be incorporated into DNA. Here, we investigate the ability of DNA polymerase β to utilize nucleotides with modified sugars. DNA polymerase β readily inserts dideoxynucleoside triphosphates but inserts ribonucleotides nearly 4 orders of magnitude less efficiently than natural deoxynucleotides. The efficiency of ribonucleotide insertion is similar to that reported for other DNA polymerases. The poor polymerase-dependent insertion represents a key step in discriminating against ribonucleotides because, once inserted, a ribonucleotide is easily extended. Likewise, a templating ribonucleotide has little effect on insertion efficiency or fidelity. In contrast to insertion and extension of a ribonucleotide, the chemotherapeutic drug arabinofuranosylcytosine triphosphate is efficiently inserted but poorly extended. These results suggest that the sugar pucker at the primer terminus plays a crucial role in DNA synthesis; a 3′-endo sugar pucker facilitates nucleotide insertion, whereas a 2′-endo conformation inhibits insertion.     

Human DNA Polymerase ? Is Able to Efficiently Extend from Multiple Consecutive Ribonucleotides

Replicative DNA polymerases (Pols) help to maintain the high fidelity of replication in large part through their strong selectivity against mispaired deoxyribonucleotides. It has recently been demonstrated that several replicative Pols from yeast have surprisingly low selectivity for deoxyribonucleotides over their analogous ribonucleotides. In human cells, ribonucleotides are found in great abundance over deoxyribonucleotides, raising the possibility that ribonucleotides are incorporated in the human genome at significant levels during normal cellular functions. To address this possibility, the ability of human DNA polymerase ϵ to incorporate ribonucleotides was tested. At physiological concentrations of nucleotides, human Pol ϵ readily inserts and extends from incorporated ribonucleotides. Almost half of inserted ribonucleotides escape proofreading by 3′ → 5′ exonuclease-proficient Pol ϵ, indicating that ribonucleotide incorporation by Pol ϵ is likely a significant event in human cells. Human Pol ϵ is also efficient at extending from primers terminating in up to five consecutive ribonucleotides. This efficient extension appears to result from reduced exonuclease activity on primers containing consecutive 3′-terminal ribonucleotides. These biochemical properties suggest that Pol ϵ is a likely source of ribonucleotides in human genomic DNA.     

Size and base composition of RNA segments in complementary strands of supercoiled plasmid ColE1 DNA

Preparations of ColEl plasmid DNA synthesized in the presence of chloramphenicol were separated into samples having gaps resulting from removal of ribonucleotides in one or the other of the complementary DNA strands. These samples were used as templates for repair resynthesis reactions using DNA polymerase of Rous sarcoma virus and α-³²P-labeled deoxyribonucleoside 5′-triphosphates. Reactions involved the incorporation of each labeled nucleotide in the presence of three unlabeled nucleotides, and also the incorporation of all four labeled nucleotides followed by complete digestion and electrophoretic separation of the products. By these two methods the RNA integrated in the light strand of ColEl DNA was found to comprise an average of 38 ribonucleotides with a base composition of 17G, 5A, 8C, and 8U. The RNA segment in the heavy strand consists of an average of 15 ribonucleotides with a base composition of 5G, 2A, 4C, and 4U.     

Solution structure of the Dickerson DNA dodecamer containing a single ribonucleotide

Ribonucleotides are frequently incorporated into DNA during replication. They are recognized and processed by several cellular enzymes, and their continued presence in the yeast nuclear genome results in replicative stress and genome instability. Thus, it is important to understand the effects of isolated ribonucleotide incorporation on DNA structure. With this goal in mind, we describe the nuclear magnetic resonance structure of the self-complementary Dickerson dodecamer sequence [d(CGC)rGd(AATTCGCG)](2) containing two symmetrically positioned riboguanosines. The absence of an observable H(1)-H(2) scalar coupling interaction indicates a C3'-endo conformation for the ribose. Longer-range structural perturbations resulting from the presence of the ribonucleotide are limited to the adjacent and transhelical nucleotides, while the global B-form DNA structure is maintained. Because crystallographic studies have indicated that isolated ribonucleotides promote global B → A transitions, we also performed molecular modeling analyses to evaluate the structural consequences of higher ribonucleotide substitution levels. Increasing the ribonucleotide content increased the minor groove width toward values more similar to that of A-DNA, but even 50% ribonucleotide substitution did not fully convert the B-DNA to A-DNA. Comparing our structure with the structure of an RNase H2-bound DNA supports the conclusion that, as with other DNA-protein complexes, the DNA conformation is strongly influenced by the interaction with the protein.     

Replication of cauliflower mosaic virus DNA in leaves and suspension culture protoplasts of cotton

Cauliflower mosaic virus (CaMV) replicated in protoplasts and in inoculated leaves of the non-host, cotton (Gossypium hirsutum, L.). Protoplasts prepared from suspension-cultured cotton cells were infected by incubation with liposome-encapsulated CaMV virions. During a 1-week culture period the amount of CaMV nucleic acid as detected by nucleic acid hybridization in the protoplasts increased significantly regardless of whether or not the protoplasts contained vacuoles. In leaves inoculated with CaMV virions or CaMV DNA, viral DNA sequences were found by leaf skeleton hybridization to be located in small circular areas. DNA extracted from ultracentrifugal pellets of homogenates of inoculated leaves contained circular, gapped CaMV DNA only when inocula contained CaMV virions, CaMV DNA, or partial nested dimer CaMV plasmid DNA. When plants had been heavily watered, the CaMV DNA recovered contained degraded CaMV DNA. The results suggest that the host range limitation for CaMV is not due to an inability to replicate or spread locally in inoculated leaves.     

Compartmentation of guanine nucleotide precursors for DNA synthesis.

We have studied the kinetics of guanine incorporation into DNA in mouse T-lymphoma (S-49) mutant cells [PNPase (purine-nucleoside phosphorylase)- and HGPRTase (hypoxanthine: guanine phosphoribosyltransferase)-deficient] that are incapable of converting dGuo (deoxyguanosine) to Gua (guanine) ribonucleotides. Of the two possible pathways for an exogenous guanine source to reach DNA, firstly: dGuo----dGMP----dGDP----dGTP and secondly: Gua----GMP----GDP----dGDP----dGTP only the second pathway was found to be functional in providing guanine for DNA replication, although deoxyguanosine readily produced toxic cellular dGTP levels via the first pathway. The functional guanine-nucleotide-precursor pools for DNA are rather small; further, the depletion of the small GMP pool, but not that of GDP, GTP and dGTP, correlated well with the inhibition of DNA synthesis by mycophenolic acid, an IMP dehydrogenase inhibitor. These results support the hypothesis that guanine-nucleotide incorporation into DNA is highly compartmentalized and that a small functional guanine-nucleotide pool, e.g., the GMP pool, may serve a crucial role in limiting the availability of DNA precursor substrate.     

Characteristics of the nascent and non-nascent small DNA molecules found in Escherichia coli

We have purified nascent DNA molecules from Escherichia coli pulse-labeled with 5-bromo[6-3H]deoxyuridine by repeated chromatography on nitrocellulose and isopycnic centrifugation in CsCl. The nascent molecules were labeled with 32P either at their 5' ends using polynucleotide kinase or at their 3' ends using terminal transferase. Compared to the non-nascent DNA of normal density, the nascent dense DNA contained a higher proportion of molecules terminated at their 5' ends with ribonucleotides. Exposure of the dense DNA to alkali generated 5' OH termini quantitatively equivalent to the number of molecules bearing 5' ribonucleotides. Experiments designed (1) to detect structures at the 5' ends of phosphatase-treated nascent DNA molecules that caused them to be resistant to hydrolysis by spleen exonuclease or (2) to detect polypeptides that were associated covalently with small DNA molecules and could be iodinated with the Bolton-Hunter reagent did not yield positive results. We conclude that many, if not all, of the intermediates in E. coli DNA replication are initiated with one or more ribonucleotides. The nascent molecules are outnumbered by small non-nascent DNA molecules in the cell, many of which appear to become slightly longer when cells are pulsed with thymidine. Many of the non-nascent DNA molecules behave as if they were self-complementary or crosslinked.     

Localization of the ribonucleotide sites in rat liver mitochondrial deoxyribonucleic acid.

Supercoiled rat liver mitochondrial DNA is relaxed by treatment with ribonucleases A, T1 or H. All the supercoiled mitochondrial DNA is sensitive to ribonuclease H and ribonuclease A, but only 35% of the supercoiled population is sensitive to ribonuclease T1. Removal of the ribonucleotides with calf thymus ribonuclease H, followed by denaturation of the mitochondrial DNA and analysis of the single-strand fragment lengths in the electron microscope, showed that the ribonucleotides were randomly located on both strands of the DNA. Endonuclease-S1 digestion of mitochondrial DNA after removal of the ribonucleotides reveals that no unique fragments are produced and ribonucleotides are randomly distributed with respect to one another. The average number of ribonucleotide sites per molecule was estimated to be between 8 and 13. Two possible mechanisms for the origin of ribonucleotide sites are discussed.     

Mechanism of mitochondrial DNA replication in mouse L-cells: localization and sequence of the light-strand origin of replication

In the novel replication mechanism of closed circular mouse L-cell mitochondrial DNA synthesis one strand of the duplex (the heavy-strand) is initiated at a defined origin and proceeds unidirectionally. Synthesis of the complementary light-strand is initiated at a different origin, located approximately two-thirds genome length from the heavy-strand origin, and also proceeds unidirectionally. The initiation of light-strand synthesis does not occur until synthesis of the heavy-strand has extended past the light-strand origin region. One intriguing consequence of this asynchrony is that the heavy-strand origin functions in a DNA duplex, while the light-strand origin functions as a single-stranded template. In order to obtain the precise location of the light-strand origin we have isolated replicative molecules in which light-strand synthesis has begun and subjected them to digestion by a combination of the single-strand specific nuclease S₁ and various restriction cndonucleases. By comparison of the sizes of the duplex fragments thus generated with those produced by cleavage of non-replicating molecules cleaved with the same enzymes we have located the 5′-end of daughter light-strands at a position 55 to 90 nucleotides from a HpaI cleavage site 0.67 genome length from the heavy-strand origin. The nucleotide sequence of a 318-base region surrounding this site, determined by chemical sequencing techniques, possesses the symmetry required for the formation of three hairpin loops. The most striking of these has a stem consisting of 12 consecutive basepairs and a 13-base loop. In the heavy-strand template, this loop contains 11 consecutive thymidine nucleotides. This light-strand origin region has been found to possess a remarkable degree of homology with several other prokaryotic and eukaryotic origin-related sequences, particularly those of the øX174 A region and the simian virus 40 EcoRII G fragment.It has previously been shown that mouse mitochondrial DNA contains alkali-labile sites, which are presumably due to the presence of ribonucleotides incorporated into the DNA. A cluster of sites, representing eight adjacent ribonucleotides, has been located in mature light strands at or near the origin of light-strand synthesis. The retention of ribonucleotides at this specific location may reflect inefficient removal of an RNA primer at the light-strand origin.     

Polymerase mu is a DNA-directed DNA/RNA polymerase

DNA polymerases are defined as such because they use deoxynucleotides instead of ribonucleotides with high specificity. We show here that polymerase mu (pol mu), implicated in the nonhomologous end-joining pathway for repair of DNA double-strand breaks, incorporates both ribonucleotides and deoxynucleotides in a template-directed manner. pol mu has an approximately 1,000-fold-reduced ability to discriminate against ribonucleotides compared to that of the related pol beta, although pol mu's substrate specificity is similar to that of pol beta in most other respects. Moreover, pol mu more frequently incorporates ribonucleotides when presented with nucleotide concentrations that approximate cellular pools. We therefore addressed the impact of ribonucleotide incorporation on the activities of factors required for double-strand break repair by nonhomologous end joining. We determined that the ligase required for this pathway readily joined strand breaks with terminal ribonucleotides. Most significantly, pol mu frequently introduced ribonucleotides into the repair junctions of an in vitro nonhomologous end-joining reaction, an activity that would be expected to have important consequences in the context of cellular double-strand break repair.     

Genomic 5-methylcytosine determination by 32P-postlabeling analysis

A simple and sensitive method for the quantitation of 5-methyldeoxycytidine in DNA has been developed by the adaptation of the Randerath 32P-postlabeling technique. Nucleic acids were digested to 3'-monophosphate nucleotides, which were converted to 32P-labeled 3',5'-bisphosphate nucleotides, the 3'-phosphate was cleaved by the action of nuclease P1, and the resultant 5'-[32P]-monophosphate nucleotides were separated by two-dimensional thin-layer chromatography. Less than 1 microgram of DNA was required for the precise quantitation of 5-methyldeoxycytidine content to a detectable limit of 0.01% of the total cytidine residues methylated. The genomic 5-methyldeoxycytidine content may thus be quantitated in tissue samples, small or selective cell populations, senescing or terminally differentiating cells, or DNA from any source. We report here, for the first time, the genomic 5-methyldeoxycytidine content of normal human bronchial epithelial and normal human pulmonary mesothelial cells. The chromatographic separation of all of the normal and some of the rare monophosphate deoxyribonucleotides and ribonucleotides has been characterized. Thus, 5-bromodeoxyuridine and the RNA contamination of DNA or the DNA contamination of RNA can also be quantitated during the same analysis.     

Different sequence elements are required for function of the cauliflower mosaic virus polyadenylation site in Saccharomyces cerevisiae compared with in plants.

We show that the polyadenylation site derived from the plant cauliflower mosaic virus (CaMV) is specifically functional in the yeast Saccharomyces cerevisiae. The mRNA 3' endpoints were mapped at the same position in yeast cells as in plants, and the CaMV polyadenylation site was recognized in an orientation-dependent manner. Mutational analysis of the CaMV 3'-end-formation signal revealed that multiple elements are essential for proper activity in yeast cells, including two upstream elements that are situated more than 100 and 43 to 51 nucleotides upstream of the poly(A) addition site and the sequences at or near the poly(A) addition site. A comparison of the sequence elements that are essential for proper function of the CaMV signal in yeast cells and plants showed that both organisms require a distal and a proximal upstream element but that these sequence elements are not identical in yeast cells and plants. The key element for functioning of the CaMV signal in yeast cells is the sequence TAGTATGTA, which is similar to a sequence previously proposed to act in yeast cells as a bipartite signal, namely, TAG ... TATGTA. Deletion of this sequence in the CaMV polyadenylation signal abolished 3'-end formation in yeast cells, and a single point mutation in this motif reduced the activity of the CaMV signal to below 15%. These results indicate that the bipartite sequence element acts as a signal for 3'-end formation in yeast cells but only together with other cis-acting elements.     

DNA sequencing by partial ribosubstitution

A new rapid method for DNA sequence analysis has been devised. In this method, base-specific cleavage is achieved at partially substituted ribonucleotides which are introduced by DNA polymerase extension in the presence of Mn2⁺. Access to a target sequence and label incorporation are achieved by extending a restriction fragment primer with DNA polymerase I. After a short initial incorporation with [α-³²P]deoxynucleotide triphosphates to label the 5′ region of the target sequence, the triphosphates are removed and the reaction mixture is divided four ways for a second primed extension. The second extension is a cold chase in the presence of Mn²⁺, all four deoxynucleotides and one of the four ribonucleotides under conditions that result in about 2% ribonucleotides substitution at each position. After cleavage at the restriction site and alkali cleavage at the positions of partial ribosubstitution, each reaction mixture is analysed by electrophoresis on a high-resolution denaturing acrylamide gel. As in the other rapid DNA sequencing methods the extent of DNA sequence that can be determined from a single experiment is limited only by the resolution of the analysing gels. At present some 100 nucleotides of sequence can be determined from a single priming reaction.