Homologous pairing in vitro initiated by DNA synthesis

A number of models have been proposed for the initiation of general genetic recombination. One of these, originally proposed by Meselson and Radding, imagines that the single-stranded 5' tail that results from strand displacement DNA repair synthesis can initiate homologous recombination by invading a homologous duplex. The resultant D-loop intermediate is then processed into mature products. We demonstrate here that an in vitro system composed of the bacteriophage T4 uvsX protein (a RecA-like "strand transferase") and part of the T4 DNA polymerase holoenzyme efficiently mediates pairing between nicked double-stranded circular and linear duplex DNAs, thereby demonstrating the feasibility of a key step in the Meselson-Radding model.     

Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro

The human Rad51 protein is essential for DNA repair by homologous recombination. In addition to Rad51 protein, five paralogs have been identified: Rad51B/Rad51L1, Rad51C/Rad51L2, Rad51D/Rad51L3, XRCC2, and XRCC3. To further characterize a subset of these proteins, recombinant Rad51, Rad51B-(His)(6), and Rad51C proteins were individually expressed employing the baculovirus system, and each was purified from Sf9 insect cells. Evidence from nickel-nitrilotriacetic acid pull-down experiments demonstrates a highly stable Rad51B.Rad51C heterodimer, which interacts weakly with Rad51. Rad51B and Rad51C proteins were found to bind single- and double-stranded DNA and to preferentially bind 3'-end-tailed double-stranded DNA. The ability to bind DNA was elevated with mixed Rad51 and Rad51C, as well as with mixed Rad51B and Rad51C, compared with that of the individual protein. In addition, both Rad51B and Rad51C exhibit DNA-stimulated ATPase activity. Rad51C displays an ATP-independent apparent DNA strand exchange activity, whereas Rad51B shows no such activity; this apparent strand exchange ability results actually from a duplex DNA destabilization capability of Rad51C. By analogy to the yeast Rad55 and Rad57, our results suggest that Rad51B and Rad51C function through interactions with the human Rad51 recombinase and play a crucial role in the homologous recombinational repair pathway.     

In vitro characterization of repair synthesis initiated by T4 endonuclease V on a synthetic DNA substrate.

The size of the repair patch produced by E. coli DNA polymerase (Pol I) following the removal of a pyrimidine dimer from DNA in response to the nicking activity of T4 endonuclease (T4 endo V) was determined. A 48-bp DNA containing a pyrimidine dimer at a defined location was labelled in the damaged strand and incubated with T4 endo V and E. coli endonuclease IV. Subsequently, DNA synthesis by DNA Pol I was carried out in the presence of four dNTPs, ATP and DNA ligase. Analysis of the reaction products on a sequencing gel revealed a ladder of only 4-oligonucleotides, 1-4 nucleotides greater in length than the fragment generated by the combined nicking activities of T4 endo V and E. coli endonuclease IV. Thus we conclude that the in vitro repair patch size of T4 endo V is 4 nucleotides and that in some cases the repaired DNA is not ligated.     

Microinjection of Escherichia coli UvrA, B, C and D proteins into fibroblasts of xeroderma pigmentosum complementation groups A and C does not result in restoration of UV-induced unscheduled DNA synthesis

The UV-induced unscheduled DNA synthesis (UDS) in cultured human fibroblasts of repair-deficient xeroderma pigmentosum complementation groups A and C was assayed after injection of identical activities of either Uvr excinuclease (UvrA, B, C and D) from Escherichia coli or endonuclease V from phage T4. Under conditions where the T4 enzyme was able to induce repair synthesis in both XP complementation groups in agreement with earlier observations (de Jonge et al., 1985), no effect of the UvrABCD excinuclease could be observed either when the enzymatic complex was injected into the cytoplasm, or when it was delivered directly into the nucleus. In addition, no effect of the E. coli excinuclease was found on the repair ability of normal repair-proficient human fibroblasts. We conclude that the UvrABCD excinuclease may not work on DNA lesions in human chromatin.     

DNA repair synthesis during base excision repair in vitro is catalyzed by DNA polymerase epsilon and is influenced by DNA polymerases alpha and delta in Saccharomyces cerevisiae.

Base excision repair is an important mechanism for correcting DNA damage produced by many physical and chemical agents. We have examined the effects of the REV3 gene and the DNA polymerase genes POL1, POL2, and POL3 of Saccharomyces cerevisiae on DNA repair synthesis is nuclear extracts. Deletional inactivation of REV3 did not affect repair synthesis in the base excision repair pathway. Repair synthesis in nuclear extracts of pol1, pol2, and pol3 temperature-sensitive mutants was normal at permissive temperatures. However, repair synthesis in pol2 nuclear extracts was defective at the restrictive temperature of 37 degrees C and could be complemented by the addition of purified yeast DNA polymerase epsilon. Repair synthesis in pol1 nuclear extracts was proficient at the restrictive temperature unless DNA polymerase alpha was inactivated prior to the initiation of DNA repair. Thermal inactivation of DNA polymerase delta in pol3 nuclear extracts enhanced DNA repair synthesis approximately 2-fold, an effect which could be specifically reversed by the addition of purified yeast DNA polymerase delta to the extract. These results demonstrate that DNA repair synthesis in the yeast base excision repair pathway is catalyzed by DNA polymerase epsilon but is apparently modulated by the presence of DNA polymerases alpha and delta.     

In vitro cleavage of double- and single-stranded DNA by plasmid RSF1010-encoded mobilization proteins.

We have used purified RSF1010 mobilization proteins to reproduce in vitro a strand-specific nicking at the plasmid origin of transfer, oriT. In the presence of Mg2+, the proteins MobA (78-kDa form of RSF1010 DNA primase), MobB, and MobC and supercoiled or linear duplex oriT DNA form large amounts of a cleavage complex, which is characterized by its sensitivity to protein-denaturant treatment. Upon addition of SDS to such a complex, a single strand break is generated in the DNA, and MobA is found linked to the 5' nick terminus, presumably covalently. The double-strand nicking activity of MobA requires, in addition to Mg2+, the presence of MobC and is stimulated by the presence of MobB. The nick site has been shown by DNA sequencing to lie at the position cleaved in vivo during transfer, between nucleotides 3138/3139 in the r strand of RSF1010. We have found that MobA will also cleave DNA at sites other than oriT if the DNA is present in single-stranded form. Breakage in this case occurs in the absence of denaturing conditions, and after prolonged incubation, reclosure can be demonstrated.     

DNA synthesis is initiated at two positions within the origin of replication of plasmid R1162.

DNA synthesis of broad host-range plasmid R1162 is initiated from two positions, flanking a large (40 bp stem, 40 bp loop) inverted repeat. Each start-point is located within a highly conserved, but oppositely oriented, 10 base-pair sequence. Synthesis from the two positions converges within the intervening inverted repeat. An analysis of deletions suggests that both start positions must be present for synthesis. A model describing possible early events in replication of plasmid R1162 is presented.     

Escherichia coli DNA synthesis in vitro: insensitivity of ATP-dependent DNA repair to inhibition by novobiocin.

Novobiocin, an effective inhibitor of DNA replicaion in Escherichia coli, is shown to have no effect on the ATP-dependent DNA repair carried out by toluenized cells after ultraviolet irradiation. Therefore novobiocin can be considered a selective inhibitor of replicative DNA synthesis in vitro.     

Strand opening by the UvrA(2)B complex allows dynamic recognition of DNA damage.

Repair proteins alter the local DNA structure during nucleotide excision repair (NER). However, the precise role of DNA melting remains unknown. A series of DNA substrates containing a unique site-specific BPDE-guanine adduct in a region of non-complementary bases were examined for incision by the Escherichia coli UvrBC endonuclease in the presence or absence of UvrA. UvrBC formed a pre-incision intermediate with a DNA substrate containing a 6-base bubble structure with 2 unpaired bases 5' and 3 unpaired bases 3' to the adduct. Formation of this bubble served as a dynamic recognition step in damage processing. UvrB or UvrBC may form one of three stable repair intermediates with DNA substrates, depending upon the state of the DNA surrounding the modified base. The dual incisions were strongly determined by the distance between the adduct and the double-stranded-single-stranded DNA junction of the bubble, and required homologous double-stranded DNA at both incision sites. Remarkably, in the absence of UvrA, UvrBC nuclease can make both 3' and 5' incisions on substrates with bubbles of 3-6 nucleotides, and an uncoupled 5' incision on bubbles of >/=>/=10 nucleotides. These data support the hypothesis that the E.coli and human NER systems recognize and process DNA damage in a highly conserved manner.     

In vitro breakage of plasmid DNA by mutagens and pesticides.

Covalently closed circular molecules of the colicinogenic plasmid E1 can serve as sensitive indicators for detecting in vitro breakage of DNA. After these molecules are radioactively labeled and purified by cesium chloride density-gradient centrifugation, they are incubated with the compounds to be tested. Samples are analyzed on alkaline sucrose gradients to determine the fraction of unbroken molecules and a breakage rate is calculated. Positive results were obtained for all three mutagenic alkylating agents (MMS, EMS, and MNNG) and of the 11 pesticides tested, dexon, dichlorvos, malathion, and methyl parathion induced breaks in molecules at a rate significantly greater than the controls.     

Limitations of the in vitro repair synthesis assay for probing the role of DNA repair in platinum resistance.

Several studies have implicated enhanced DNA repair in acquired platinum resistance. To better understand the mechanism of increased repair we have employed an in vitro assay using cell-free extracts from platinum sensitive and resistant murine and human cell lines. Since the platinum resistant murine cell lines used in our previous studies had shown increased repair of diaminocyclohexane(dach)-Pt-DNA adducts while one of the resistant human cell lines did not, we have measured in vitro repair synthesis on DNA damaged by (d,l)-trans-1,2-diaminocyclohexanedichloroplatinum(II) (PtCl2(dach)). The results of this assay were strongly dependent on the method used to calculate repair synthesis activity and appeared to disagree with previous estimates of repair activity in these cell lines. By one method of calculation the in vitro repair synthesis assay underestimated the ratio of repair activities in the resistant versus the sensitive murine cell lines, while by the other method the in vitro assay overestimated the ratio of repair activities in the resistant versus the sensitive human cell lines.     

Evaluating metabolic stress and plasmid stability in plasmid DNA production by Escherichia coli

In the context of recombinant DNA technology, the development of feasible and high-yielding plasmid DNA production processes has regained attention as more evidence for its efficacy as vectors for gene therapy and DNA vaccination arise. When producing plasmid DNA in Escherichia coli, a number of biological restraints, triggered by plasmid maintenance and replication as well as culture conditions are responsible for limiting final biomass and product yields. This termed "metabolic burden" can also cause detrimental effects on plasmid stability and quality, since the cell machinery is no longer capable of maintaining an active metabolism towards plasmid synthesis and the stress responses elicited by plasmid maintenance can also cause increased plasmid instability. The optimization of plasmid DNA production bioprocesses is still hindered by the lack of information on the host metabolic responses as well as information on plasmid instability. Therefore, systematic and on-line approaches are required not only to characterise this "metabolic burden" and plasmid stability but also for the design of appropriate metabolic engineering and culture strategies. The monitoring tools described to date rapidly evolve from laborious, off-line and at-line monitoring to online monitoring, at a time-scale that enables researchers to solve these bioprocessing problems as they occur. This review highlights major E. coli biological alterations caused by plasmid maintenance and replication, possible causes for plasmid instability and discusses the ability of currently employed bioprocess monitoring techniques to provide information in order to circumvent metabolic burden and plasmid instability, pointing out the possible evolution of these methods towards online bioprocess monitoring.     

Fluid shear stress enhanced DNA synthesis in cultured endothelial cells during repair of mechanical denudation

We have previously observed a stimulatory effect of fluid shear stress on the regeneration of cultured endothelial cell layers after mechanical denudation. In this study we examined how fluid shear stress affects endothelial cell DNA synthesis during regeneration. Following mechanical denudation of narrow linear areas, monolayers of bovine aortic endothelial cells cultured on plastic dishes were subjected to shear stress of 1.3-4.1 dynes/cm2 for 24-48 hours in a specially designed apparatus. After the application of shear stress, cells were stained with propidium iodide, and its fluorescence intensity, reflecting cellular DNA content, was measured using photometric fluorescence microscopy. The DNA content of cells exposed to shear stress increased significantly more than that of paired, static control cells (p less than 0.005 to p less than 0.001). The DNA histogram showed that cells exposed to shear stress contained a relatively high proportion of cells located in the S, G2, and M phases of the cell cycle as compared with the static control. These data suggest that fluid shear stress enhances endothelial cell DNA synthesis during the repair of mechanical denudation.     

Sequential binding of DNA repair proteins RPA and ERCC1 to XPA in vitro.

Recent studies have shown that many proteins are involved in the early steps of nucleotide excision repair and that there are some interactions between nucleotide excision repair proteins, suggesting that these interactions are important in the reaction mechanism. The xeroderma pigmentosum group A protein (XPA) was shown to bind to the replication protein A (RPA) or the excision repair cross complementing rodent repair deficiency group 1 protein (ERCC1), and these interactions might be involved in the damage-recognition and/or incision steps, of nucleotide excision repair. Here we show that the XPA regions required for the binding to the 70 and 34 kDa subunits of RPA are located in the middle and on N-terminal regions of XPA, respectively. These regions do not overlap with the ERCC1-binding region of XPA, and a ternary protein complex of RPA, XPA and ERCC1 was detected in vitro. In addition, using the surface plasmon resonance biosensor, the binding of RPA and ERCC1 to XPA was investigated. The dissociation constants (KD) of RPA and ERCC1 with XPA were 1.9 x 10(-8 )and 2.5 x 10(-7) M, respectively. Moreover, our results suggest the sequential binding of RPA and ERCC1 to XPA.     

Manganese as a mutagenic agent during in vitro DNA synthesis.

The effect of Mn²⁺, a known mutagen, on the fidelity of DNA synthesis $\text{in vitro}$ by avian myeloblastosis DNA polymerase has been determined. Substitution of Mn²⁺ for Mg²⁺ leads to an enhanced incorporation of noncomplementary deoxynucleotides as well as complementary ribonucleotides with either poly (A) or poly (C) as templates. Since this polymerase lacks any detectable deoxyribonuclease activity, the $\text{in vitro}$ mutagenic effect of Mn²⁺ in promoting errors in base-pairing does not result from any diminished proof-reading function.     

Conserved domains in DNA repair proteins and evolution of repair systems.

A detailed analysis of protein domains involved in DNA repair was performed by comparing the sequences of the repair proteins from two well-studied model organisms, the bacterium Escherichia coli and yeast Saccharomyces cerevisiae, to the entire sets of protein sequences encoded in completely sequenced genomes of bacteria, archaea and eukaryotes. Previously uncharacterized conserved domains involved in repair were identified, namely four families of nucleases and a family of eukaryotic repair proteins related to the proliferating cell nuclear antigen. In addition, a number of previously undetected occurrences of known conserved domains were detected; for example, a modified helix-hairpin-helix nucleic acid-binding domain in archaeal and eukaryotic RecA homologs. There is a limited repertoire of conserved domains, primarily ATPases and nucleases, nucleic acid-binding domains and adaptor (protein-protein interaction) domains that comprise the repair machinery in all cells, but very few of the repair proteins are represented by orthologs with conserved domain architecture across the three superkingdoms of life. Both the external environment of an organism and the internal environment of the cell, such as the chromatin superstructure in eukaryotes, seem to have a profound effect on the layout of the repair systems. Another factor that apparently has made a major contribution to the composition of the repair machinery is horizontal gene transfer, particularly the invasion of eukaryotic genomes by organellar genes, but also a number of likely transfer events between bacteria and archaea. Several additional general trends in the evolution of repair proteins were noticed; in particular, multiple, independent fusions of helicase and nuclease domains, and independent inactivation of enzymatic domains that apparently retain adaptor or regulatory functions.