Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes

Nucleotide excision DNA repair is mechanistically conserved across all kingdoms of life. In prokaryotes, this multi-enzyme process requires six proteins: UvrA?D, DNA polymerase I and DNA ligase. To examine how UvrC locates the UvrB? DNA pre-incision complex at a site of damage, we have labeled UvrB and UvrC with different colored quantum dots and quantitatively observed their interactions with DNA tightropes under a variety of solution conditions using oblique angle fluorescence imaging. Alone, UvrC predominantly interacts statically with DNA at low salt. Surprisingly, however, UvrC and UvrB together in solution bind to form the previously unseen UvrBC complex on duplex DNA. This UvrBC complex is highly motile and engages in unbiased one-dimensional diffusion. To test whether UvrB makes direct contact with the DNA in the UvrBC?DNA complex, we investigated three UvrB mutants: Y96A, a b-hairpin deletion and D338N. These mutants affected the motile properties of the UvrBC complex, indicating that UvrB is in intimate contact with the DNA when bound to UvrC. Given the in vivo excess of UvrB and the abundance of UvrBC in our experiments, this newly identified complex is likely to be the predominant form of UvrC in the cell. © 2013 The Author(s)

Theoretical and Practical Design Approach of Wireless Power Systems

The paper introduces the main issues concerned with the conceptual design process of wireless power systems. It analyses the electromagnetic design of the inductive magnetic coupler and proposes the key formulas to optimize its electrical parameters for a particular load. For this purpose, a very detailed analysis is given focusing on the mathematical concept procedure for determination of the key factors influencing proper coupling coils design. It also suggests basic topologies for conceptual design of power electronics and discusses its proper connection to the grid. The proposed design strategy is verified by experimental laboratory measurement including analyses of leakage magnetic field

Repair activity of base and nucleotide excision repair enzymes for guanine lesions induced by nitrosative stress

Nitric oxide (NO) induces deamination of guanine, yielding xanthine and oxanine (Oxa). Furthermore, Oxa reacts with polyamines and DNA binding proteins to form cross-link adducts. Thus, it is of interest how these lesions are processed by DNA repair enzymes in view of the genotoxic mechanism of NO. In the present study, we have examined the repair capacity for Oxa and Oxa–spermine cross-link adducts (Oxa–Sp) of enzymes involved in base excision repair (BER) and nucleotide excision repair (NER) to delineate the repair mechanism of nitrosative damage to guanine. Oligonucleotide substrates containing Oxa and Oxa–Sp were incubated with purified BER and NER enzymes or cell-free extracts (CFEs), and the damage-excising or DNA-incising activity was compared with that for control (physiological) substrates. The Oxa-excising activities of Escherichia coli and human DNA glycosylases and HeLa CFEs were 0.2–9% relative to control substrates, implying poor processing of Oxa by BER. In contrast, DNA containing Oxa–Sp was incised efficiently by UvrABC nuclease and SOS-induced E.coli CFEs, suggesting a role of NER in ameliorating genotoxic effects associated with nitrosative stress. Analyses of the activity of CFEs from NER-proficient and NER-deficient human cells on Oxa–Sp DNA confirmed further the involvement of NER in the repair of nitrosative DNA damage

Crystal Structure of the FeS Cluster–Containing Nucleotide Excision Repair Helicase XPD

DNA damage recognition by the nucleotide excision repair pathway requires an initial step identifying helical distortions in the DNA and a proofreading step verifying the presence of a lesion. This proofreading step is accomplished in eukaryotes by the TFIIH complex. The critical damage recognition component of TFIIH is the XPD protein, a DNA helicase that unwinds DNA and identifies the damage. Here, we describe the crystal structure of an archaeal XPD protein with high sequence identity to the human XPD protein that reveals how the structural helicase framework is combined with additional elements for strand separation and DNA scanning. Two RecA-like helicase domains are complemented by a 4Fe4S cluster domain, which has been implicated in damage recognition, and an α-helical domain. The first helicase domain together with the helical and 4Fe4S-cluster–containing domains form a central hole with a diameter sufficient in size to allow passage of a single stranded DNA. Based on our results, we suggest a model of how DNA is bound to the XPD protein, and can rationalize several of the mutations in the human XPD gene that lead to one of three severe diseases, xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy

Removal of Misincorporated Ribonucleotides from Prokaryotic Genomes: An Unexpected Role for Nucleotide Excision Repair

Stringent steric exclusion mechanisms limit the misincorporation of ribonucleotides by high-fidelity DNA polymerases into genomic DNA. In contrast, low-fidelity Escherichia coli DNA polymerase V (pol V) has relatively poor sugar discrimination and frequently misincorporates ribonucleotides. Substitution of a steric gate tyrosine residue with alanine (umuC_Y11A) reduces sugar selectivity further and allows pol V to readily misincorporate ribonucleotides as easily as deoxynucleotides, whilst leaving its poor base-substitution fidelity essentially unchanged. However, the mutability of cells expressing the steric gate pol V mutant is very low due to efficient repair mechanisms that are triggered by the misincorporated rNMPs. Comparison of the mutation frequency between strains expressing wild-type and mutant pol V therefore allows us to identify pathways specifically directed at ribonucleotide excision repair (RER). We previously demonstrated that rNMPs incorporated by umuC_Y11A are efficiently removed from DNA in a repair pathway initiated by RNase HII. Using the same approach, we show here that mismatch repair and base excision repair play minimal back-up roles in RER in vivo. In contrast, in the absence of functional RNase HII, umuC_Y11A-dependent mutagenesis increases significantly in ΔuvrA, uvrB5 and ΔuvrC strains, suggesting that rNMPs misincorporated into DNA are actively repaired by nucleotide excision repair (NER) in vivo. Participation of NER in RER was confirmed by reconstituting ribonucleotide-dependent NER in vitro. We show that UvrABC nuclease-catalyzed incisions are readily made on DNA templates containing one, two, or five rNMPs and that the reactions are stimulated by the presence of mispaired bases. Similar to NER of DNA lesions, excision of rNMPs proceeds through dual incisions made at the 8th phosphodiester bond 5′ and 4th-5th phosphodiester bonds 3′ of the ribonucleotide. Ribonucleotides misinserted into DNA can therefore be added to the broad list of helix-distorting modifications that are substrates for NER

Crystal structure of the UvrB dimer: insights into the nature and functioning of the UvrAB damage engagement and UvrB-DNA complexes

UvrB has a central role in the highly conserved UvrABC pathway functioning not only as a damage recognition element but also as an essential component of the lesion tracking machinery. While it has been recently confirmed that the tracking assembly comprises a UvrA2B2 heterotetramer, the configurations of the damage engagement and UvrB-DNA handover complexes remain obscure. Here, we present the first crystal structure of a UvrB dimer whose biological significance has been verified using both chemical cross-linking and electron paramagnetic resonance spectroscopy. We demonstrate that this dimeric species stably associates with UvrA and forms a UvrA2B2-DNA complex. Our studies also illustrate how signals are transduced between the ATP and DNA binding sites to generate the helicase activity pivotal to handover and formation of the UvrB2-DNA complex, providing key insights into the configurations of these important repair intermediates

STUDIES ON THE BIOSYNTHESIS OF THE HYPERMODIFIED BASE IN BACTERIOPHAGE SP15 DEOXYRIBONUCLEIC ACID.

Abstract not availabl

Preleukemic Fusion Genes Induced via Ionizing Radiation

Although the prevalence of leukemia is increasing, the agents responsible for this increase are not definitely known. While ionizing radiation (IR) was classified as a group one carcinogen by the IARC, the IR-induced cancers, including leukemia, are indistinguishable from those that are caused by other factors, so the risk estimation relies on epidemiological data. Several epidemiological studies on atomic bomb survivors and persons undergoing IR exposure during medical investigations or radiotherapy showed an association between radiation and leukemia. IR is also known to induce chromosomal translocations. Specific chromosomal translocations resulting in preleukemic fusion genes (PFGs) are generally accepted to be the first hit in the onset of many leukemias. Several studies indicated that incidence of PFGs in healthy newborns is up to 100-times higher than childhood leukemia with the same chromosomal aberrations. Because of this fact, it has been suggested that PFGs are not able to induce leukemia alone, but secondary mutations are necessary. PFGs also have to occur in specific cell populations of hematopoetic stem cells with higher leukemogenic potential. In this review, we describe the connection between IR, PFGs, and cancer, focusing on recurrent PFGs where an association with IR has been established

Novel and recurrent germline alterations in the MLH1 and MSH2 genes identified in hereditary nonpolyposis colorectal cancer patients in Slovakia

Hereditary non-polyposis colorectal cancer (HNPCC) is associated with germline mutations in DNA mismatch repair genes, predominantly MSH2 and MLH1. Mutation carriers develop cancers in the colorectum, endometrium, ovary, stomach, small intestine and the upper urinary tract. We describe here the results of a mutational analysis of 11 unrelated HNPCC patients by direct genomic sequencing of MLH1 and MSH2. The alterations found include 7 novel changes and 4 different pathogenic mutations described previously in Poland, Moldavia, Finland, Germany, France and USA. Four novel pathogenic mutations in the MLH1 gene include two frameshift mutations (c.1150delG and c.1210_1211delCT), one missense mutation (c.793C>A) and one intron-exon border mutation (c.546- 2A>C). The last change resulted in the skipping of exon 7, as shown by sequencing of RT-PCR products. The only novel MSH2 pathogenic change was a nonsense mutation c.1129C>T. The novel intronic change c.381-41A>G in MLH1 was found in a patient carrying a previously-described mutation in the MSH2 gene. Interestingly, two unrelated patients carried also a novel change in the promoter region of MLH1 in one of the CpG islands (c.-269C>G). However, this alteration does not abrogate transcription, as shown by RT-PCR analysis. In summary, most (approximately 80%) pathogenic germline mutations detected in the studied group of patients by direct genomic sequencing of MLH1 and MSH2 were located in the MLH1 gene. These and previous data indicate that the majority of germline point mutations and small deletions/insertions in HNPCC families in Slovakia affect the MLH1 locus