A type IB topoisomerase with DNA repair activities

Previously we have characterized type IB DNA topoisomerase V (topo V) in the hyperthermophile Methanopyrus kandleri. The enzyme has a powerful topoisomerase activity and is abundant in M. kandleri. Here we report two characterizations of topo V. First, we found that its N-terminal domain has sequence homology with both eukaryotic type IB topoisomerases and the integrase family of tyrosine recombinases. The C-terminal part of the sequence includes 12 repeats, each repeat consisting of two similar but distinct helix-hairpin-helix motifs; the same arrangement is seen in recombination protein RuvA and mammalian DNA polymerase β. Second, on the basis of sequence homology between topo V and polymerase β, we predict and demonstrate that topo V possesses apurinic/apyrimidinic (AP) site-processing activities that are important in base excision DNA repair: (i) it incises the phosphodiester backbone at the AP site, and (ii) at the AP endonuclease cleaved AP site, it removes the 5′ 2-deoxyribose 5-phosphate moiety so that a single-nucleotide gap with a 3′-hydroxyl and 5′-phosphate can be filled by a DNA polymerase. Topo V is thus the prototype for a new subfamily of type IB topoisomerases and is the first example of a topoisomerase with associated DNA repair activities.

Managing DNA polymerases: Coordinating DNA replication, DNA repair, and DNA recombination

Two important and timely questions with respect to DNA replication, DNA recombination, and DNA repair are: (i) what controls which DNA polymerase gains access to a particular primer-terminus, and (ii) what determines whether a DNA polymerase hands off its DNA substrate to either a different DNA polymerase or to a different protein(s) for the completion of the specific biological process? These questions have taken on added importance in light of the fact that the number of known template-dependent DNA polymerases in both eukaryotes and in prokaryotes has grown tremendously in the past two years. Most notably, the current list now includes a completely new family of enzymes that are capable of replicating imperfect DNA templates. This UmuC-DinB-Rad30-Rev1 superfamily of DNA polymerases has members in all three kingdoms of life. Members of this family have recently received a great deal of attention due to the roles they play in translesion DNA synthesis (TLS), the potentially mutagenic replication over DNA lesions that act as potent blocks to continued replication catalyzed by replicative DNA polymerases. Here, we have attempted to summarize our current understanding of the regulation of action of DNA polymerases with respect to their roles in DNA replication, TLS, DNA repair, DNA recombination, and cell cycle progression. In particular, we discuss these issues in the context of the Gram-negative bacterium, Escherichia coli, that contains a DNA polymerase (Pol V) known to participate in most, if not all, of these processes.

Roles of DNA polymerases V and II in SOS-induced error-prone and error-free repair in Escherichia coli

DNA polymerase V, composed of a heterotrimer of the DNA damage-inducible UmuC and UmuD\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{^{\prime}}}}\end{equation*}\end{document} proteins, working in conjunction with RecA, single-stranded DNA (ssDNA)-binding protein (SSB), β sliding clamp, and γ clamp loading complex, are responsible for most SOS lesion-targeted mutations in Escherichia coli, by catalyzing translesion synthesis (TLS). DNA polymerase II, the product of the damage-inducible polB (dinA ) gene plays a pivotal role in replication-restart, a process that bypasses DNA damage in an error-free manner. Replication-restart takes place almost immediately after the DNA is damaged (≈2 min post-UV irradiation), whereas TLS occurs after pol V is induced ≈50 min later. We discuss recent data for pol V-catalyzed TLS and pol II-catalyzed replication-restart. Specific roles during TLS for pol V and each of its accessory factors have been recently determined. Although the precise molecular mechanism of pol II-dependent replication-restart remains to be elucidated, it has recently been shown to operate in conjunction with RecFOR and PriA proteins.

Tuning DNA “strings”: Modulating the rate of DNA replication with mechanical tension

Recent experiments have measured the rate of replication of DNA catalyzed by a single enzyme moving along a stretched template strand. The dependence on tension was interpreted as evidence that T7 and related DNA polymerases convert two (n = 2) or more single-stranded template bases to double helix geometry in the polymerization site during each catalytic cycle. However, we find structural data on the T7 enzyme–template complex indicate n = 1. We also present a model for the “tuning” of replication rate by mechanical tension. This model considers only local interactions in the neighborhood of the enzyme, unlike previous models that use stretching curves for the entire polymer chain. Our results, with n = 1, reconcile force-dependent replication rate studies with structural data on DNA polymerase complexes.

Somatic mosaicism in Wiskott–Aldrich syndrome suggests in vivo reversion by a DNA slippage mechanism

Somatic mosaicism caused by in vivo reversion of inherited mutations has been described in several human genetic disorders. Back mutations resulting in restoration of wild-type sequences and second-site mutations leading to compensatory changes have been shown in mosaic individuals. In most cases, however, the precise genetic mechanisms underlying the reversion events have remained unclear, except for the few instances where crossing over or gene conversion have been demonstrated. Here, we report a patient affected with Wiskott–Aldrich syndrome (WAS) caused by a 6-bp insertion (ACGAGG) in the WAS protein gene, which abrogates protein expression. Somatic mosaicism was documented in this patient whose majority of T lymphocytes expressed nearly normal levels of WAS protein. These lymphocytes were found to lack the deleterious mutation and showed a selective growth advantage in vivo. Analysis of the sequence surrounding the mutation site showed that the 6-bp insertion followed a tandem repeat of the same six nucleotides. These findings strongly suggest that DNA polymerase slippage was the cause of the original germ-line insertion mutation in this family and that the same mechanism was responsible for its deletion in one of the propositus T cell progenitors, thus leading to reversion mosaicism.

Requirement of proliferating cell nuclear antigen in RAD6-dependent postreplicational DNA repair.

The proliferating cell nuclear antigen (PCNA) acts as a processivity factor for replicative DNA polymerases and is essential for DNA replication. In vitro studies have suggested a role for PCNA-in the repair synthesis step of nucleotide excision repair, and PCNA interacts with the cyclin-dependent kinase inhibitor p21. However, because of the lack of genetic evidence, it is not clear which of the DNA repair processes are in fact affected by PCNA in vivo. Here, we describe a PCNA mutation, pol30-46, that confers ultraviolet (UV) sensitivity but has no effect on growth or cell cycle progression, and the mutant pcna interacts normally with DNA polymerase delta and epsilon. Genetic studies indicate that the pol30-46 mutation is specifically defective in RAD6-dependent postreplicational repair of UV damaged DNA, and this mutation impairs the error-free mode of bypass repair. These results implicate a role for PCNA as an intermediary between DNA replication and postreplicational DNA repair.

Cloning of thermostable DNA polymerases from hyperthermophilic marine Archaea with emphasis on Thermococcus sp. 9 degrees N-7 and mutations affecting 3'-5' exonuclease activity.

Five extremely thermophilic Archaea from hydrothermal vents were isolated, and their DNA polymerases were cloned and expressed in Escherichia coli. Protein splicing elements (inteins) are present in many archaeal DNA polymerases, but only the DNA polymerase from strain GB-C contained an intein. Of the five cloned DNA polymerases, the Thermococcus sp. 9 degrees N-7 DNA polymerase was chosen for biochemical characterization. Thermococcus sp. 9 degrees N-7 DNA polymerase exhibited temperature-sensitive strand displacement activity and apparent Km values for DNA and dNTP similar to those of Thermococcus litoralis DNA polymerase. Six substitutions in the 3'-5' exonuclease motif I were constructed in an attempt to reduce the 3'-5' exonuclease activity of Thermococcus sp. 9 degrees N-7 DNA polymerase. Five mutants resulted in no detectable 3'-5' exonuclease activity, while one mutant (Glul43Asp) had <1% of wild-type activity.

Sequence-specific arrest of primer extension on single-stranded DNA by an oligonucleotide-minor groove binder conjugate.

A minor groove binder (MGB) derivative (N-3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate tripeptide; CDPI3) was covalently linked to the 5' or 3' end of several oligodeoxyribonucleotides (ODNs) totally complementary or possessing a single mismatch to M13mp19 single-stranded DNA. Absorption thermal denaturation and slot-blot hybridization studies showed that conjugation of CDPI3 to these ODNs increased both the specificity and the strength with which they hybridized. Primer extension of the same phage DNA by a modified form of phage T7 DNA polymerase (Sequenase) was physically blocked when a complementary 16-mer with a conjugated 5'-CDPI3 moiety was hybridized to a downstream site. Approximately 50% of the replicating complexes were arrested when the blocking ODN was equimolar to the phage DNA. Inhibition was unaffected by 3'-capping of the ODN with a hexanol group or by elimination of a preannealing step. Blockage was abolished when a single mismatch was introduced into the ODN or when the MGB was either removed or replaced by a 5'-acridine group. A 16-mer with a 3'-CDPI3 moiety failed to arrest primer extension, as did an unmodified 32-mer. We attribute the exceptional stability of hybrids formed by ODNs conjugated to a CDPI3 to the tethered tripeptide binding in the minor groove of the hybrid. When that group is linked to the 5' end of a hybridized ODN, it probably blocks DNA synthesis by inhibiting strand displacement. These ODNs conjugated to CDPI3 offer attractive features as diagnostic probes and antigene agents.

Heterogeneity of primer extension products in asymmetric PCR is due both to cleavage by a structure-specific exo/endonuclease activity of DNA polymerases and to premature stops.

In PCR, DNA polymerases from thermophilic bacteria catalyze the extension of primers annealed to templates as well as the structure-specific cleavage of the products of primer extension. Here we show that cleavage by Thermus aquaticus and Thermus thermophilus DNA polymerases can be precise and substantial: it occurs at the base of the stem-loop structure assumed by the single strand products of primer extension using as template a common genetic element, the promoter-operator of the Escherichia coli lactose operon, and may involve up to 30% of the products. The cleavage is independent of primer, template, and triphosphates, is dependent on substrate length and temperature, requires free ends and Mg2+, and is absent in DNA polymerases lacking the 5'-->3' exonuclease, such as the Stoffel fragment and the T7 DNA polymerase. Heterogeneity of the extension products results also from premature detachment of the enzyme approaching the 5' end of the template.

Mutagenesis by third-strand-directed psoralen adducts in repair-deficient human cells: high frequency and altered spectrum in a xeroderma pigmentosum variant.

Psoralen-conjugated triple-helix-forming oligonucleotides have been used to generate site-specific mutations within mammalian cells. To investigate factors influencing the efficiency of oligonucleotide-mediated gene targeting, the processing of third-strand-directed psoralen adducts was compared in normal and repair-deficient human cells. An unusually high mutation frequency and an altered mutation pattern were seen in xeroderma pigmentosum variant (XPV) cells compared with normal, xeroderma pigmentosum group A (XPA), and Fanconi anemia cells. In XPV, targeted mutations were produced in the supF reporter gene carried in a simian virus 40 vector at a frequency of 30%, 3-fold above that in normal or Fanconi anemia cells and 6-fold above that in XPA. The mutations generated by targeted psoralen crosslinks and monoadducts in the XPV cells formed a pattern distinct from that in the other three cell lines, with mutations occurring not just at the damaged site but also at adjacent base pairs. Hence, the XPV cells may have an abnormality in trans-lesion bypass synthesis during repair and/or replication, implicating a DNA polymerase or an accessory factor as a basis of the defect in XPV. These results may help to elucidate the repair deficiency in XPV, and they raise the possibility that genetic manipulation via triplex-targeted mutagenesis may be enhanced by modulation of the XPV-associated activity in normal cells.

Reconstitution of repair-gap UV mutagenesis with purified proteins from Escherichia coli: a role for DNA polymerases III and II.

Using a cell-free system for UV mutagenesis, we have previously demonstrated the existence of a mutagenic pathway associated with nucleotide-excision repair gaps. Here, we report that this pathway can be reconstituted by using six purified proteins: UvrA, UvrB, UvrC, DNA helicase II, DNA polymerase III core, and DNA ligase. This establishes the minimal requirements for repair-gap UV mutagenesis. DNA polymerase II could replace DNA polymerase III, although less effectively, whereas DNA polymerase I, the major repair polymerase, could not. DNA sequence analysis of mutations generated in the in vitro reaction revealed a spectrum typical of mutations targeted to UV lesions. These observations suggest that repair-gap UV mutagenesis is performed by DNA polymerase III, and to a lesser extent by DNA polymerase II, by filling-in of a rare class of excision gaps that contain UV lesions.

Catalytic editing properties of DNA polymerases.

Enzymatic incorporation of 2',3'-dideoxynucleotides into DNA results in chain termination. We report that 3'-esterified 2'-deoxynucleoside 5'-triphosphates (dNTPs) are false chain-terminator substrates since DNA polymerases, including human immunodeficiency virus reverse transcriptase, can incorporate them into DNA and, subsequently, use this new 3' end to insert the next correctly paired dNTP. Likewise, a DNA substrate with a primer chemically esterified at the 3' position can be extended efficiently upon incubation with dNTPs and T7 DNA polymerase lacking 3'-to-5' exonuclease activity. This enzyme is also able to use dTTP-bearing reporter groups in the 3' position conjugated through amide or thiourea bonds and cleave them to restore a DNA chain terminated by an amino group at the 3' end. Hence, a number of DNA polymerases exhibit wide catalytic versatility at the 3' end of the nascent DNA strand. As part of the polymerization mechanism, these capabilities extend the number of enzymatic activities associated with these enzymes and also the study of interactions between DNA polymerases and nucleotide analogues.

Genomic DNA fingerprinting by restriction fragment end labeling.

A typing method for bacteria was developed and applied to several species, including Escherichia coli and Actinobacillus actinomycetemcomitans. Total genomic DNA was digested with a restriction endonuclease, and fragments were enabled with [alpha-32P]dATP by using the Klenow fragment of DNA polymerase and separated by electrophoresis in 6% polyacrylamide/8 M urea (sequencing gel). Depending on the restriction endonuclease and the bacterium, the method produced approximately 30-50 well-separated fragments in the size range of 100-400 nucleotides. For A. actinomycetemcomitans, all strains had bands in common. Nevertheless, many polymorphisms could be observed, and the 31 strains tested could be classified into 29 distinct types. Furthermore, serotype-specific fragments could be assigned for the three serotypes investigated. The method described is very sensitive, allowing more distinct types to be distinguished than other commonly used typing methods. When the method was applied to 10 other clinically relevant bacterial species, both species-specific bands and strain-specific bands were found. Isolates from different locations of one patient showed indistinguishable patterns. Computer-assisted analysis of the DNA fingerprints allowed the determination of similarity coefficients. It is concluded that genomic fingerprinting by restriction fragment end labeling (RFEL) is a powerful and generally applicable technique to type bacterial species.

Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis.

Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of the label. These primers carry a fluorescein derivative at the 5' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within the primer sequence as acceptors. Adjustment of the donor-acceptor spacing through the placement of the modified thymidine in the primer sequence allowed generation of four primers, all having strong absorption at a common excitation wavelength (488 nm) and fluorescence emission maxima of 525, 555, 580, and 605 nm. The ET efficiency of these primers ranges from 65% to 97%, and they exhibit similar electrophoretic mobilities by gel electrophoresis. With argon-ion laser excitation, the fluorescence of the ET primers and of the DNA sequencing fragments generated with ET primers is 2- to 6-fold greater than that of the corresponding primers or fragments labeled with single dyes. The higher fluorescence intensity of the ET primers allows DNA sequencing with one-fourth of the DNA template typically required when using T7 DNA polymerase. With single-stranded M13mp18 DNA as the template, a typical sequencing reaction with ET primers on a commercial sequencer provided DNA sequences with 99.8% accuracy in the first 500 bases. ET primers should be generally useful in the development of other multiplex DNA sequencing and analysis methods.

NMR of enzymatically synthesized uniformly 13C15N-labeled DNA oligonucleotides.

A procedure for the enzymatic synthesis of uniformly 13C15N-labeled DNA oligonucleotides in milligram quantities for NMR studies is described. Deoxynucleotides obtained from microorganisms grown on 13C and 15N nutrient sources are enzymatically phosphorylated to dNTPs, and the dNTPs are incorporated into oligonucleotides using a 3'-5' exonuclease-deficient mutant of Klenow fragment of DNA polymerase I and an oligonucleotide template primer designed for efficient separation of labeled product DNA from unlabeled template. The labeling strategy has been used to uniformly label one or the other oligonucleotide strand in the DNA duplex dGGCAAAACGG.dCCGTTTTGCC in order to facilitate assignment and structure determination by NMR. Application of 15N and 13C heteronuclear NMR experiments to isotopically labeled DNA is presented.