Locating the uracil-5-yl radical formed upon photoirradiation of 5-bromouracil-substituted DNA

In a previous study, we found that 2-deoxyribonolactone is effectively generated in the specific 5-bromouracil (^BrU)-substituted sequence 5′-(G/C)[A]_{n = 1,2}^BrU^BrU-3′ and proposed that a formed uracil-5-yl radical mainly abstracts the C1′ hydrogen from the 5′-side of ^BrU^BrU under 302-nm irradiation condition. In the present work, we performed photoirradiation of ^BrU-substituted DNA in the presence of a hydrogen donor, tetrahydrofuran, to quench the uracil-5-yl radical to uracil and then subjected the sample to uracil DNA glycosylase digestion. Slab gel sequence analysis indicated that uracil residues were formed at the hot-spot sequence of 5′-(G/C)[A]_{n = 1,2}^BrU^BrU-3′ in 302-nm irradiation of ^BrU-substituted DNA. Furthermore, we found that the uracil residue was also formed at the reverse sequence 5′-^BrU^BrU[A]_{n = 1,2}(G/C)-3′, which suggests that both 5′-(G/C)[A]_{n = 1,2}^BrU^BrU-3′ and 5′-^BrU^BrU[A]_{n = 1,2}(G/C)-3′ are hot-spot sequences for the formation of the uracil-5-yl radical.     

Enhancement of recA protein-promoted DNA strand exchange activity by volume-occupying agents.

To investigate the in vivo effects of macromolecular crowding we examined the effect of inert macromolecules such as polyvinyl alcohol and polyethylene glycol on the in vitro activity of recA protein. The addition of either of these volume-occupying agents enables recA protein to promote homologous pairing and exchange of DNA strands at an otherwise nonpermissive magnesium ion concentration. In the presence of these macromolecules, both the rate of recA protein association with single-stranded DNA (ssDNA) and the steady-state affinity of recA protein for ssDNA are increased. Consequently, the ability of recA protein to compete with ssDNA-binding protein (SSB protein) is enhanced, and the inhibitory effects of SSB protein on the formation of recA protein-ssDNA presynaptic complexes are eliminated. Because the ability of recA protein to bind to ssDNA-containing secondary structures is also enhanced in volume-occupied solution, joint molecule formation is not greatly reduced when SSB protein is omitted from the reaction. Thus, increased recA protein interactions with ssDNA contribute to enhanced presynaptic complex formation. In addition, polyvinyl alcohol and polyethylene glycol must also affect another property of recA protein, i.e. self-association, which is required for synapsis and DNA strand exchange. Our examination of DNA strand exchange in the presence of volume-occupying agents helps to reconcile the requirement for elevated magnesium ion concentrations in recA protein-promoted recombination reactions in vitro, with a presumably low magnesium ion concentration in vivo.     

Effect of neocarzinostatin-induced strand scission on the template activity of DNA for DNA polymerase I.

Neocarzinostatin (NCS), an antitumor protein antibiotic that causes strand scissions of DNA both in vitro and in vivo, is shown to lower the template activity of DNA for DNA polymerase Iin vitro. There is a correlation between the extent of strand scission and the degree of inhibition, maximal inhibition of the polymerase reaction being obtained under conditions promoting maximal strand scission. These effects can be related to the concentrations of NCS and of 2-mercaptoethanol and are maximized by pretreatment of the DNA with drug. Results from polymerase assays in which the amount of drug-treated DNA template was varied at a constant level of the enzyme suggest that the sites associated with NCS-induced breaks are nonfunctional in DNA synthesis but bind DNA polymerase I. The binding of the enzyme to the inactive sites is further confirmed using [203 Hg] polymerase. It is shown that the lowering of the template activity of DNA by NCS under conditions of strand scission is due to the generation of a large number of inactive sites that block, competitively, the binding of DNA polymerase to the active sites on the template. Furthermore, the inhibition of DNA synthesis, which depends on the extent of strand breakage and on the relative amounts of template and enzyme, can be reversed by increasing the levels of template or polymerase. The finding that DNA synthesis directed by poly [d(A-T)] is much more sensitive to NCS than that primed by poly [d(G-C)] suggests that the drug preferentially interacts at regions containing adenine and/or thymine residues.     

DNA strand scission and ADP-ribosyltransferase activity during murine erythroleukemia cell differentiation

The accumulation of DNA strand breaks and activation of ADP-ribosyltransferase (ADPRT) have recently been associated with cellular differentiation. Murine erythroleukemia (MEL) cells undergo erythropoietic differentiation when exposed to dimethyl sulfoxide (Me2SO) and several studies have suggested that DNA strand scission induced by this agent is a prerequisite for expression of the differentiated phenotype. Me2SO induction of MEL cells has also been associated with increases in ADPRT activity in one study, but not in another. We have monitored the effects of Me2SO on DNA strand breaks in preformed and replicating MEL cell DNA. The results clearly demonstrate that DNA fragmentation is not detectable during Me2SO induction of MEL differentiation, even in the presence of 3-aminobenzamide, an inhibitor of ADPRT. Further, these results are consistent with an absence of detectable changes in both endogenous and total potential ADPRT activity during Me2SO-induced MEL differentiation. These findings would argue against Me2SO induction of DNA strand scission and ADPRT in MEL cells undergoing differentiation.     

Ligation activity of FLP recombinase. The strand ligation activity of a site-specific recombinase using an activated DNA substrate.

The FLP protein of the 2-microns plasmid of yeast belongs to the integrase family of site-specific recombinases whose members form a covalent bond between a conserved tyrosine of the recombinase and the 3'-phosphoryl group at the site of cleavage. We have made an activated DNA substrate and have shown that FLP can promote efficient strand ligation without forming a covalent intermediate with the DNA substrate. The strand ligation activity of FLP is independent of its ability to cleave DNA. Since site-specific recombinases are members of the larger class of topoisomerases, these findings may be generally applicable to other members of this class of enzymes.     

Sequencing double-stranded DNA by strand displacement.

Duplex probes with five base single-stranded overhangs can capture dsDNA targets from type IIS restriction nuclease digests. Ligation generates a predesigned nick site, where DNA polymerase can generate sequencing ladders by strand displacement or nick translation in the presence of trace amounts of dideoxynucleotides. This allows dsDNA targets to be captured from mixtures and directly sequenced without subcloning, purification or denaturation.     

Replisome fate upon encountering a leading strand block and clearance from DNA by recombination proteins

Replication forks that collapse upon encountering a leading strand lesion are reactivated by a recombinative repair process called replication restart. Using rolling circle DNA substrates to model replication forks, we examine the fate of the helicase and both DNA polymerases when the leading strand polymerase is blocked. We find that the helicase continues over 0.5 kb but less than 3 kb and that the lagging strand DNA polymerase remains active despite its connection to a stalled leading strand enzyme. Furthermore, the blocked leading strand polymerase remains stably bound to the replication fork, implying that it must be dismantled from DNA in order for replication restart to initiate. Genetic studies have identified at least four gene products required for replication restart, RecF, RecO, RecR, and RecA. We find here that these proteins displace a stalled polymerase at a DNA template lesion. Implications of these results for replication fork collapse and recovery are discussed.     

RecA protein-facilitated DNA strand breaks. A mechanism for bypassing DNA structural barriers during strand exchange

RecA protein promotes an unexpectedly efficient DNA strand exchange between circular single-stranded DNA and duplex DNAs containing short (50-400-base pair) heterologous sequences at the 5' (initiating) end. The major mechanism by which this topological barrier is bypassed involves DNA strand breakage. Breakage is both strand and position specific, occurring almost exclusively in the displaced (+) strand of the duplex within a 15-base pair region of the heterology/homology junction. Breakage also requires recA protein, ATP hydrolysis, and homologous sequences 3' to the heterology. Although the location of the breaks and the observed requirements clearly indicate a major role for recA protein in this phenomenon, the molecular mechanism is not yet clear. The breakage may reflect a DNA structure and/or some form of structural stress within the DNA during recA protein-mediated DNA pairing which either exposes the DNA at this precise position to the action of a contaminating nuclease or induces a direct mechanical break. We also find that when heterology is located at the 3' end of the linear duplex, strand exchange is halted (without DNA breakage) about 500 base pairs from the homology/heterology junction.     

DNA damaging activity of ellagic acid derivatives

A strain of yeast rendered repair deficient by the conditional expression of the RAD52 locus was used to search for natural products capable of damaging DNA. Four ellagic acid derivatives, namely 3,3'-dimethyl-4'-O-beta-D-glucopyranosyl ellagic acid (1), 3,3',4-trimethyl-4'-O-beta-D-glucopyranosyl ellagic acid (2), 3'-methyl-3,4-O,O-methylidene ellagic acid (3) and 3'-methyl-3,4-O,O-methylidene-4'-O-beta-D-glucopyranosyl ellagic acid (4), were identified by this assay as DNA damaging natural principles from several plants, including Alangium javanicum, Anisophyllea apetala, Crypteronia paniculata, Mouririi sp. and Scholtzia parviflora. Although none of the isolated principles mediated frank strand scission of DNA in vitro, all of them potently inhibited the growth of yeast in the absence of expression of RAD52.     

Preparation and melting of single strand circular DNA loops.

A method for preparation of single strand DNA circles of almost arbitrary sequence is described. By ligating two sticky ended hairpins together a linear duplex is formed, closed at both ends by single stranded loops. The melting characteristics of such loops are investigated using optical absorbance and NMR. It is shown by comparison with the corresponding linear sequence (closed circle minus the end loops) that the effects of end fraying and the strand concentration dependence of the melting temperature are eliminated in the circular form. Over the concentration range examined (0.5 to 2.0 micromolar strands), the circular DNA has a monophasic melting curve, while the linear duplex is biphasic, probably due to hairpin formation. Since effects of duplex to single strands dissociation do not contribute to melting of the circular molecules (dumbells), these DNAs present a realistic experimental model for examining local thermal stability in DNA.     

Qualitative and quantitative aspects of intercalator-induced DNA strand breaks.

The intercalating agents, adriamycin and ellipticine, were previously found to produce DNA strand breaks associated with DNA-protein crosslinks in mouse leukemia L1210 cells. The current work explores the nature of the agents that produce this effect and the quantitative relationship between the breaks and crosslinks. The protein-associated DNA breaks were produced by a wide variety of intercalators in addition to the above-mentioned compounds: actinomycin D, daunoycin, ethidium and lucanthone (miracil D). Treatment with several drugs that bind to DNA without intercalation, or that inhibit DNA synthesis without binding to DNA, did not cause DNA breaks. The strand break and crosslink frequencies were quantitated by means of alkaline elution methods. The strand break and crosslink frequencies were found to be within a factor of 2 of each other over a range of concentrations of adriamycin and ellipticine. It is proposed that intercalation-induced distortion of the DNA helix leads to strand scission by a nuclease which becomes bound to one terminus of the break so as to form a DNA-protein crosslink.     

HIV-1 DNA integration: mechanism of viral DNA cleavage and DNA strand transfer.

Retroviral DNA integration involves a coordinated set of DNA cutting and joining reactions. Linear viral DNA is cleaved at each 3' end to generate the precursor ends for integration. The resulting recessed 3' ends are inserted into target DNA by a subsequent DNA strand transfer reaction. Purified HIV-1 integration protein carries out both of these steps in vitro. Two novel forms of the dinucleotide cleaved from HIV-1 DNA were identified and one, a cyclic dinucleotide, was used to analyze the stereochemical course of viral DNA cleavage. Both viral DNA cleavage and DNA strand transfer display inversion at chiral phosphorothioates during the course of the reaction. These results suggest that both reactions occur by a one-step mechanism without involvement of a covalent protein-DNA intermediate.     

pBR322 plasmid DNA modified with 2-acetylaminofluorene derivatives: transforming activity and in vitro strand cleavage by the Escherichia coli uvrABC endonuclease

Covalently closed circular plasmid DNA was treated with three reactive derivatives of 2-acetylaminofluorene: N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF), its 7-iodo derivative (N-Aco- AAIF ) and N-hydroxy-N-2-aminofluorene (N-OH-AF), and tested as substrates for the Escherichia coli uvrABC endonuclease and for transformation frequencies on wild-type, uvrA, recA, uvrArecA and polA mutant strains. The uvrABC endonuclease reacted with all three substrates with high efficiency, implicating this enzyme in the repair of DNA containing all three types of adducts. However, only AAF- and AAIF -DNA showed greatly reduced survival on uvrA mutants (five adducts/lethal hit) relative to wild-type (20 adducts/lethal hit). AF-DNA survived equally well on uvrA mutant and wild-type cells, and at a much higher level of modification (60 adducts/lethal hit). A mutation in recA had only a minor effect on the survival of either DNA. The polA mutation reduced the survival of the AAF-treated DNA to the same extent as the uvrA mutation (five adducts/lethal hit). Also AF-DNA showed reduced survival on polA mutant cells versus wild-type. However, many more adducts (20/lethal hit) were tolerated than for AAF-DNA, indicating that AF lesions in the template do not efficiently block replication of DNA.     

DNA strand break repair and neurodegeneration

A number of DNA repair disorders are known to cause neurological problems. These disorders can be broadly characterised into early developmental, mid-to-late developmental or progressive. The exact developmental processes that are affected can influence disease pathology, with symptoms ranging from early embryonic lethality to late-onset ataxia. The category these diseases belong to depends on the frequency of lesions arising in the brain, the role of the defective repair pathway, and the nature of the mutation within the patient. Using observations from patients and transgenic mice, we discuss the importance of double strand break repair during neuroprogenitor proliferation and brain development and the repair of single stranded lesions in neuronal function and maintenance.     

DNA strand displacement, strand annealing and strand swapping by the Drosophila Bloom's syndrome helicase

Genetic analysis of the Drosophila Bloom's syndrome helicase homolog (mus309/DmBLM) indicates that DmBLM is required for the synthesis-dependent strand annealing (SDSA) pathway of homologous recombination. Here we report the first biochemical study of DmBLM. Recombinant, epitope-tagged DmBLM was expressed in Drosophila cell culture and highly purified protein was prepared from nuclear extracts. Purified DmBLM exists exclusively as a high molecular weight (~1.17 MDa) species, is a DNA-dependent ATPase, has 3′→5′ DNA helicase activity, prefers forked substrate DNAs and anneals complementary DNAs. High-affinity DNA binding is ATP-dependent and low-affinity ATP-independent interactions contribute to forked substrate DNA binding and drive strand annealing. DmBLM combines DNA strand displacement with DNA strand annealing to catalyze the displacement of one DNA strand while annealing a second complementary DNA strand.     

An overview of homologous pairing and DNA strand exchange proteins.

Processes fundamental to all models of genetic recombination include the homologous pairing and subsequent exchange of DNA strands. Biochemical analysis of these events has been conducted primarily on the recA protein of Escherichia coli, although proteins which can promote such reactions have been purified from many sources, both prokaryotic and eukaryotic. The activities of these homologous pairing and DNA strand exchange proteins are either ATP-dependent, as predicted based on the recA protein paradigm, or, more unexpectedly, ATP-independent. This review examines the reactions promoted by both classes of proteins and highlights their similarities and differences. The mechanistic implications of the apparent existence of 2 classes of strand exchange protein are discussed.     

The ATPase activity of RecA is needed to push the DNA strand exchange through heterologous regions.

The role of ATP hydrolysis during the RecA-mediated recombination reaction is addressed in this paper. Recent studies indicated that the RecA-promoted DNA strand exchange between completely homologous double- and single-stranded DNA can be very efficient in the absence of ATP hydrolysis. In this work we demonstrate that the energy derived from the ATP hydrolysis is strictly needed to drive the DNA strand exchange through the regions where the interacting DNA molecules are not in a homologous register. Therefore, in addition to the role of the ATP hydrolysis in promoting the dissociation of RecA from the products of the recombination reaction, as described earlier, ATP hydrolysis also plays a crucial role in the actual process of strand exchange, provided that the lack of homologous register obstructs the process of branch migration.     

Weak strand displacement activity enables human DNA polymerase beta to expand CAG/CTG triplet repeats at strand breaks

Using synthetic DNA constructs in vitro, we find that human DNA polymerase beta effectively catalyzes CAG/CTG triplet repeat expansions by slippage initiated at nicks or 1-base gaps within short (14 triplet) repeat tracts in DNA duplexes under physiological conditions. In the same constructs, Escherichia coli DNA polymerase I Klenow Fragment exo(-) is much less effective in expanding repeats, because its much stronger strand displacement activity inhibits slippage by enabling rapid extension through two downstream repeats into flanking non-repeat sequence. Polymerase beta expansions of CAG/CTG repeats, observed over a 32-min period at rates of approximately 1 triplet added per min, reveal significant effects of break type (nick versus gap), strand composition (CTG versus CAG), and dNTP substrate concentration, on repeat expansions at strand breaks. At physiological substrate concentrations (1-10 microm of each dNTP), polymerase beta expands triplet repeats with the help of weak strand displacement limited to the two downstream triplet repeats in our constructs. Such weak strand displacement activity in DNA repair at strand breaks may enable short tracts of repeats to be converted into longer, increasingly mutable ones associated with neurological diseases.