Combined loss of three DNA damage response pathways renders C. elegans intolerant to light

Infliction of DNA damage initiates a complex cellular reaction – the DNA damage response – that involves both signaling and DNA repair networks with many redundancies and parallel pathways. Here, we reveal the three strategies that the simple multicellular eukaryote, C. elegans, uses to deal with DNA damage induced by light. Separately inactivating repair or replicative bypass of photo-lesions results in cellular hypersensitivity towards UV-light, but impeding repair of replication associated DNA breaks does not. Yet, we observe an unprecedented synergistic relationship when these pathways are inactivated in combination. C. elegans mutants that lack nucleotide excision repair (NER), translesion synthesis (TLS) and alternative end joining (altEJ) grow undisturbed in the dark, but become sterile when grown in light. Even exposure to very low levels of normal daylight impedes animal growth. We show that NER and TLS operate to suppress the formation of lethal DNA breaks that require polymerase theta-mediated end joining (TMEJ) for their repair. Our data testifies to the enormous genotoxicity of light and to the demand of multiple layers of protection against an environmental threat that is so common.     

NEIL3-Dependent Regulation of Cardiac Fibroblast Proliferation Prevents Myocardial Rupture

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Structural Insights into the Mechanism of Base Excision by MBD4

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.     

DNA ADP-Ribosylation Stalls Replication and Is Reversed by RecF-Mediated Homologous Recombination and Nucleotide Excision Repair

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Senescence and N release from clover roots following permanent excision of the shoot

Six to seven week old red clover plants (Trifolium pratense L. cv Merviot) were used to investigate the time-course of root senescence following complete and permanent excision from the shoot. Plants were grown in sand culture watered with nutrient solution. After excision of the shoots, roots were left in situ and sampled over a period of up to 42 days. Respiration rate began to decrease immediately after excision, reaching 50% of its initial value after 24 h. The decline involved a reduction in the capacity of the respiratory pathways as measured in the presence of an uncoupler (FCCP) and exogenous glucose. The reduction in respiration could be prevented by supplying 100 mM sucrose to excised roots incubated in nutrient solution at the time of excision, but not 4–5 days after excision. There was a steady reduction in the protein and soluble sugar concentrations from the time of excision and a smaller reduction in starch. Free amino acid concentrations increased immediately after excision, but the temporal dynamics differed between individual amino acids. The total concentration of free amino acids rose to a maximum value 6–13 days after excision, before declining. Under these conditions roots survived for a remarkably long period of time. Depending on the experiment, cell viability, measured as the percentage of cells with positive turgor, was unchanged for at least 20 days, and complete loss of viability was not observed until 34–42 days after excision. There was no appreciable loss of N from the roots until cell viability declined significantly. The potential implications of these results for modelling and management of N cycling in cropping systems is discussed briefly.     

甲板甲床部分切除联合苯酚烧灼术治疗嵌甲症的临床研究

目的:探讨甲板甲床部分切除联合苯酚烧灼术治疗嵌甲症的临床疗效。方法:选择武汉市中心医院2004年10月至2013年5月收治的115例148趾拇趾嵌甲进行研究。以2004年10月至2007年12月的患者53例(67趾)作为对照组行单纯甲板甲床部分切除术治疗,2008年1月至2013年5月的患者62例(81趾)作为观察组行甲板甲床部分切除联合苯酚烧灼术治疗。对比两组患者的出血、疼痛缓解和康复时间,了解一年后患者复发情况及满意度。结果:148趾手术伤口均一期愈合,观察组病例术后出血时间、疼痛缓解时间(1.85±0.42天、13.25±2.17小时)较对照组(2.69±0.53天、21.54±2.56小时)低,但术后恢复时间(11.32±2.37天)较对照组(8.93±2.06天)长,差异均有统计学意义(P0.05),观察组和对照组的复发率及总体满意率分别为6.15%、12.5%及97.06%、91.07%,两组差异并无统计学意义(P0.05)。结论:甲板甲床部分切除联合苯酚烧灼术治疗嵌甲症与单纯甲板甲床部分切除术的疗效相当,但可显著减少患者痛苦。     

The contribution of Nth and Nei DNA glycosylases to mutagenesis in Mycobacterium smegmatis

The increased prevalence of drug resistant strains of Mycobacterium tuberculosis (Mtb) indicates that significant mutagenesis occurs during tuberculosis disease in humans. DNA damage by host-derived reactive oxygen/nitrogen species is hypothesized to be critical for the mutagenic process in Mtb thus, highlighting an important role for DNA repair enzymes in maintenance of genome fidelity. Formamidopyrimidine (Fpg/MutM/Fapy) and EndonucleaseVIII (Nei) constitute the Fpg/Nei family of DNA glycosylases and together with EndonucleaseIII (Nth) are central to the base excision repair pathway in bacteria. In this study we assess the contribution of Nei and Nth DNA repair enzymes in Mycobacterium smegmatis (Msm), which retains a single nth homologue and duplications of the Fpg (fpg1 and fpg2) and Nei (nei1 and nei2) homologues. Using an Escherichia coli nth deletion mutant, we confirm the functionality of the mycobacterial nth gene in the base excision repair pathway. Msm mutants lacking nei1, nei2 and nth individually or in combination did not display aberrant growth in broth culture. Deletion of nth individually results in increased UV-induced mutagenesis and combinatorial deletion with the nei homologues results in reduced survival under oxidative stress conditions and an increase in spontaneous mutagenesis to rifampicin. Deletion of nth together with the fpg homolgues did not result in any growth/survival defects or changes in mutation rate. Furthermore, no differential emergence of the common rifampicin resistance conferring genotypes were noted. Collectively, these data confirm a role for Nth in base excision repair in mycobacteria and further highlight a novel interplay between the Nth and Nei homologues in spontaneous mutagenesis.     

Human DNA polymerases catalyze lesion bypass across benzo[a]pyrene-derived DNA adduct clustered with an abasic site

The combined action of oxidative stress and genotoxic polycyclic aromatic hydrocarbons derivatives can lead to cluster-type DNA damage that includes both a modified nucleotide and a bulky lesion. As an example, we investigated the possibility of repair of an AP site located opposite a minor groove-positioned (+)-trans-BPDE-dG or a base-displaced intercalated (+)-cis-BPDE-dG adduct (BP lesion) by a BER system. Oligonucleotides with single uracil residue in the certain position were annealed with complementary oligonucleotides bearing either a cis- or trans-BP adduct. Digestion with uracil DNA glycosylase was utilized to generate an AP site which was then hydrolyzed by APE1, and the resulting gap was processed by X-family DNA polymerases β (Polβ) and λ (Polλ), or Y-family polymerase ι (Polι). By varying reaction conditions, namely, Mg²⁺/Mn²⁺ replacement/combination and ionic strength decrease, we found that under certain conditions both Polβ and Polι can catalyze lesion bypass across both cis- and trans-BP adducts in the presence of physiological dNTP concentrations. Polβ and Polι catalyze gap filling trans-lesion synthesis in an error prone manner. By contrast, Polλ selectively introduced the correct dCTP opposite the modified dG in the case of cis-BP-dG adduct only, and did not bypass the stereoisomeric trans-adduct under any of the conditions examined. The results suggest that Polλ is a specialized polymerase that can process these kinds of lesions.     

How a mismatch repair enzyme balances the needs for efficient lesion processing and minimal action on undamaged DNA

Comment on: Maiti A, et al. Proc Natl Acad Sci USA 2012; 109:8091-6.     

Ribonucleotides as nucleotide excision repair substrates

The incorporation of ribonucleotides in DNA has attracted considerable notice in recent years, since the pool of ribonucleotides can exceed that of the deoxyribonucleotides by at least 10–20-fold, and single ribonucleotide incorporation by DNA polymerases appears to be a common event. Moreover ribonucleotides are potentially mutagenic and lead to genome instability. As a consequence, errantly incorporated ribonucleotides are rapidly repaired in a process dependent upon RNase H enzymes. On the other hand, global genomic nucleotide excision repair (NER) in prokaryotes and eukaryotes removes damage caused by covalent modifications that typically distort and destabilize DNA through the production of lesions derived from bulky chemical carcinogens, such as polycyclic aromatic hydrocarbon metabolites, or via crosslinking. However, a recent study challenges this lesion-recognition paradigm. The work of Vaisman et al. (2013) [34] reveals that even a single ribonucleotide embedded in a deoxyribonucleotide duplex is recognized by the bacterial NER machinery in vitro. In their report, the authors show that spontaneous mutagenesis promoted by a steric-gate pol V mutant increases in uvrA, uvrB, or uvrC strains lacking rnhB (encoding RNase HII) and to a greater extent in an NER-deficient strain lacking both RNase HI and RNase HII. Using purified UvrA, UvrB, and UvrC proteins in in vitro assays they show that despite causing little distortion, a single ribonucleotide embedded in a DNA duplex is recognized and doubly-incised by the NER complex. We present the hypothesis to explain the recognition and/or verification of this small lesion, that the critical 2′-OH of the ribonucleotide – with its unique electrostatic and hydrogen bonding properties – may act as a signal through interactions with amino acid residues of the prokaryotic NER complex that are not possible with DNA. Such a mechanism might also be relevant if it were demonstrated that the eukaryotic NER machinery likewise incises an embedded ribonucleotide in DNA.