The regulation of DNA repair during development

DNA repair is important in such phenomena as carcinogenesis and aging. While much is known about DNA repair in single-cell systems such as bacteria, yeast, and cultured mammalian cells, it is necessary to examine DNA repair in a developmental context in order to completely understand its processes in complex metazoa such as man. We present data to support the notion that proliferating cells from organ systems, tumors, and embryos have a greater DNA repair capacity than terminally differentiated, nonproliferating cells. Differential expression of repair genes and accessibility of chromatin to repair enzymes are considered as determinants in the developmental regulation of DNA repair.     

腹腔镜下腹壁疝修补术的治疗现状与进展

腹壁疝是普外科最常见的疾病之一,主要包括腹股沟疝、切口疝、造口旁疝等,手术是其唯一的治愈方法。腹腔镜腹壁疝修 补术是在无张力疝修补术的基础上发展起来的一种微创技术,主要包括腹腔镜下腹壁疝修补术(LIHR),如经腹腔镜腹膜前补片修 补术(TAPP)和完全腹膜外补片修补术(TEP),腹腔镜下切口疝修补术及腹腔镜下造口旁疝修补术。其术后常见并发症与传统疝修 补术相同,但发生率显著低于传统的开腹疝修补术。与传统疝修补术相比,腹腔镜腹壁疝修补术具有术后疼痛轻、并发症少、疤痕 小、复发率低等优势,因此临床应用前景广阔。本文主要就腹腔镜下腹壁疝修补术的治疗现状与进展进行了综述。     

Identification of novel DNA repair proteins via primary sequence, secondary structure, and homology

Background  

DNA repair is the general term for the collection of critical mechanisms which repair many forms of DNA damage such as methylation or ionizing radiation. DNA repair has mainly been studied in experimental and clinical situations, and relatively few information-based approaches to new extracting DNA repair knowledge exist. As a first step, automatic detection of DNA repair proteins in genomes via informatics techniques is desirable; however, there are many forms of DNA repair and it is not a straightforward process to identify and classify repair proteins with a single optimal method. We perform a study of the ability of homology and machine learning-based methods to identify and classify DNA repair proteins, as well as scan vertebrate genomes for the presence of novel repair proteins. Combinations of primary sequence polypeptide frequency, secondary structure, and homology information are used as feature information for input to a Support Vector Machine (SVM).     

Visualization of repair of double-strand breaks in the bacteriophage T7 genome without normal DNA replication

An in vitro system based on extracts of Escherichia coli infected with bacteriophage T7 is able to repair double-strand breaks in a T7 genome with efficiencies of 20% or more. To achieve this high repair efficiency it is necessary that the reaction mixtures contain molecules of donor DNA that bracket the double-strand break. Gaps as long as 1,600 nucleotides are repaired almost as efficiently as simple double-strand breaks. DNA synthesis was measured while repair was taking place. It was found that the amount of DNA synthesis associated with repair of a double-strand break was below the level of detection possible with this system. Furthermore, repair efficiencies were the same with or without normal levels of T7 DNA polymerase. However, the repair required the 5'-->3' exonuclease encoded by T7 gene 6. The high efficiency of DNA repair allowed visualization of the repaired product after in vitro repair, thereby assuring that the repair took place in vitro rather than during an in vivo growth step after packaging.     

A re-evaluation of DNA repair during skeletal myogenesis in vitro

In previous studies on DNA repair during myogenesis, comparisons made of repair in post-replication myoblasts and in myotubes led to the conclusion that the capacity to repair damage in DNA decreased during myoblast differentiation. Using unscheduled DNA synthesis in response to UV-induced damage as an indicator of DNA repair in a myogenic line of rat skeletal muscle, it is demonstrated that nuclei in myotubes possess identical repair capacity as that in proliferating myoblasts. Furthermore, a brief increase in DNA repair capacity was observed to immediately follow the cessation of replicative DNA synthesis. This transient increase in repair capacity is consistent with the data of earlier reports and explains the previous but inappropriate conclusion that repair diminishes during myogenic differentiation. This transient increase in the capacity to repair DNA was not observed in a developmentally defective, non-differentiating line of similar myogenic origin.     

Repair of DNA damage in a mitochondrial lysate of Xenopus laevis oocytes.

We examined DNA repair activities of a mitochondrial lysate derived from Xenopus laevis oocytes. Plasmid DNA, exposed to HCl, H2O2 or UV light, was used as the substrate for the in vitro repair reaction. DNA synthesis in the lysate was stimulated 2-8-fold by such lesions, indicating the presence of excision repair activities. This repair DNA synthesis was not affected by aphidicolin, but was sensitive to N-ethylmaleimide. Thus the mitochondrial DNA polymerase, i.e., pol gamma is indeed involved in the reaction. Actual repair of the depurinated DNA was demonstrated by using the polymerase chain reaction (PCR), where the amount of the amplified DNA fragment increased significantly if the depurinated template was incubated in the lysate prior to the PCR. UV-irradiated DNA, on the other hand, restored its ability as a PCR template only if the repair reaction was carried out under the light. Therefore, in this system, UV-induced damage is repaired mainly by photoreactivation. These results show that mitochondria of Xenopus oocytes possess excision repair as well as photolyase activities, and that the in vitro repair system described here should be useful for further molecular characterization of such DNA repair machinery.     

New tricks for old drugs: the anticarcinogenic potential of DNA repair inhibitors

Defective or abortive repair of DNA lesions has been associated with carcinogenesis. Therefore it is imperative for a cell to accurately repair its DNA after damage if it is to return to a normal cellular phenotype. In certain circumstances, if DNA damage cannot be repaired completely and with high fidelity, it is more advantageous for an organism to have some of its more severely damaged cells die rather than survive as neoplastic transformants. A number of DNA repair inhibitors have the potential to act as anticarcinogenic compounds. These drugs are capable of modulating DNA repair, thus promoting cell death rather than repair of potentially carcinogenic DNA damage mediated by error-prone DNA repair processes. In theory, exposure to a DNA repair inhibitor during, or immediately after, carcinogenic exposure should decrease or prevent tumorigenesis. However, the ability of DNA repair inhibitors to prevent cancer development is difficult to interpret depending upon the system used and the type of genotoxic stress. Inhibitors may act on multiple aspects of DNA repair as well as the cellular signaling pathways activated in response to the initial damage. In this review, we summarize basic DNA repair mechanisms and explore the effects of a number of DNA repair inhibitors that not only potentiate DNA-damaging agents but also decrease carcinogenicity. In particular, we focus on a novel anti-tumor agent, β-lapachone, and its potential to block transformation by modulating poly(ADP-ribose) polymerase-1.     

Site-specific repair of cyclobutane pyrimidine dimers in a positioned nucleosome by photolyase and T4 endonuclease V in vitro.

Since genomic DNA is folded into nucleosomes, and DNA damage is generated all over the genome, a central question is how DNA repair enzymes access DNA lesions and how they cope with nucleosomes. To investigate this topic, we used a reconstituted nucleosome (HISAT nucleosome) as a substrate to generate DNA lesions by UV light (cyclobutane pyrimidine dimers, CPDs), and DNA photolyase and T4 endonuclease V (T4-endoV) as repair enzymes. The HISAT nucleosome is positioned precisely and contains a long polypyrimidine region which allows one to monitor formation and repair of CPDs over three helical turns. Repair by photolyase and T4-endoV was inefficient in nucleosomes compared with repair in naked DNA. However, both enzymes showed a pronounced site-specific modulation of repair on the nucleosome surface. Removal of the histone tails did not substantially enhance repair efficiency nor alter the site specificity of repair. Although photolyase and T4-endoV are different enzymes with different mechanisms, they exhibited a similar site specificity in nucleosomes. This implies that the nucleosome structure has a decisive role in DNA repair by exerting a strong constraint on damage accessibility. These findings may serve as a model for damage recognition and repair by more complex repair mechanisms in chromatin.     

Uv- and Gamma-Radiation Sensitive Mutants of Arabidopsis Thaliana

Arabidopsis seedlings repair UV-induced DNA damage via light-dependent and -independent pathways. The mechanism of the ``dark repair' pathway is still unknown. To determine the number of genes required for dark repair and to investigate the substrate-specificity of this process we isolated mutants with enhanced sensitivity to UV radiation in the absence of photoreactivating light. Seven independently derived UV sensitive mutants were isolated from an EMS-mutagenized population. These fell into six complementation groups, two of which (UVR1 and UVH1) have previously been defined. Four of these mutants are defective in the dark repair of UV-induced pyrimidine [6-4] pyrimidinone dimers. These four mutant lines are sensitive to the growth-inhibitory effects of gamma radiation, suggesting that this repair pathway is also involved in the repair of some type of gamma-induced DNA damage product. The requirement for the coordinate action of several different gene products for effective repair of pyrimidine dimers, as well as the nonspecific nature of the repair activity, is consistent with nucleotide excision repair mechanisms previously described in Saccharomyces cerevisiae and nonplant higher eukaryotes and inconsistent with substrate-specific base excision repair mechanisms found in some bacteria, bacteriophage, and fungi.     

Potentially lethal damage repair is due to the difference of DNA double-strand break repair under immediate and delayed plating conditions

Cells plated immediately after irradiation on nutrient agar (immediate plating) exhibit a lower survival than cells which are kept under nongrowth conditions before plating (delayed plating). The difference between the survival curves obtained after immediate plating and delayed plating is considered to exhibit the cell's capacity to repair potentially lethal damage. In yeast evidence has been presented previously for the DNA double-strand break (DSB) as the molecular lesion involved in the repair of potentially lethal damage observed at the cellular level. Radiation-induced DSB are repaired in cells plated on nutrient agar, i.e., under growth conditions, as well as in cells kept under nongrowth conditions. In this paper DSB repair under growth and nongrowth conditions is studied with the help of the yeast mutant rad54-3 which is temperature conditional for DSB repair. It is shown that the extent of repair of potentially lethal damage can be varied by shifting the relative fractions of repair of DSB under growth conditions versus nongrowth conditions. Repair of DSB in cells plated on nutrient agar is promoted when glucose is substituted by Na-succinate as an energy source. As a result the immediate plating survival curve approaches the delayed plating survival curve, thus reducing the operationally defined repair of potentially lethal damage. We show that this reduced potentially lethal damage repair is caused, however, by a higher amount of DSB repair in cells immediately plated on succinate agar as compared to glucose agar.     

Excision repair of UV-damaged plasmid DNA in Xenopus oocytes is mediated by DNA polymerase alpha (and/or delta).

We studied DNA repair by injecting plasmids containing random pyrimidine dimers into Xenopus oocytes. We demonstrated excision repair by recovering plasmids and analyzing them with T4 UV endonuclease treatment and alkaline agarose gel electrophoresis. The mechanism for excision repair of these plasmids appears to be processive, rather than distributive, since repair occurs in 'all or none' fashion. At less than 4-5 dimers/plasmid, nearly all repair occurs within 4-6 hours (approximately 10(10) dimers repaired per oocyte); the oocyte, therefore, has abundant repair activity. Specific antibodies and inhibitors were used to determine enzymes involved in repair. We conclude that DNA polymerase alpha (and/or delta) is required because repair is inhibited by antibodies to human DNA polymerase alpha, as well as by aphidicolin, an inhibitor of polymerases alpha (and/or delta). Repair was not inhibited by hydroxyurea, cytosine beta-D-arabinofuranoside, or inhibitors of topoisomerase II (novobiocin). Oocyte repair does not activate semi-conservative DNA replication, nor is protein synthesis required. Photoreactivation cannot account for repair because dimer removal is independent of exogenous light.     

Recombinational repair of hydrogen peroxide-induced damages in DNA of phage T4

Recently, hydrogen peroxide and its free-radical product, the hydroxyl radical (OH.) have been identified as major sources of DNA damage in living organisms. They occur as ubiquitous metabolic by-products and, in humans, cause several thousand damages in a cell's DNA per day. They are thought to be a major source of DNA damage leading to aging and cancer in multicellular organisms. This raises two questions. First, what pathways are used in repair of DNA damages caused by H2O2 and OH.? Second, a new theory has been proposed that sexual reproduction (sex) evolved to promote repair of DNA in the germ line of organisms. If this theory is correct, then the type of repair specifically available during the sexual process should be able to deal with important natural lesions such as those produced by H2O2 and OH. . Does this occur? We examined repair of hydrogen peroxide damage to DNA, using a standard bacteriophage T4 test system in which sexual reproduction is either permitted or not permitted. Post-replication recombinational repair and denV-dependent excision repair are not dependent on sex. Both of these processes had little or no effect on lethal H2O2 damage. Also, an enzyme important in repair of H2O2-induced DNA damage in the E. coli host cells, exonuclease III, was not utilized in repair of lethal H2O2 damage to the phage. However, multiplicity reactivation, a recombinational form of repair depending on the sexual interaction of two or more of the bacteriophage, was found to repair lethal H2O2 damages efficiently. Our results lend support to the repair hypothesis of sex. Also the homology-dependent recombinational repair utilized in the phage sexual process may be analogous to the homology-dependent recombination which is widespread in diploid eucaryotes. The recombinational repair pathway found in phage T4 may thus be a widely applicable model for repair of the ubiquitous DNA damage caused by endogenous oxidative reactions.     

Repair of x-ray-induced single strand breaks in toluenized Escherichia coli cells.

We have used sedimentation in alkali to estimate the repair of X-ray-induced single strand breaks in the DNA of irradiated toluenized Escherichia coli cells. Extensive repair requires no exogenous cofactors except ATP although other individual NTPs (except U) or dNTPs can substitute for ATP. There is no repair in polA or resA cells and since nicotinamide mononucleotide (NMN) inhibits repair in wild type cells we interpret the results as indicating that both ligase and polymerase I are needed for repair but that the amount of any gap filling is small and extensive repair replication is not necessary.     

The time course of repair of ultraviolet-induced DNA damage; implications for the structural organisation of repair

Alternative molecular mechanisms can be envisaged for the cellular repair of UV-damaged DNA. In the "random collision" model, DNA damage distributed throughout the genome is recognised and repaired by a process of random collision between DNA damage and repair enzymes. The other model assumes a "processive" mechanism, whereby DNA is scanned for damage by a repair complex moving steadily along its length. These two models give different predictions concerning the time course of repair. Random collision should result in a declining rate of repair with time as the concentration of lesions in the DNA falls; but the processive model predicts a constant rate of repair until scanning is complete. We have examined the time course of DNA repair in human fibroblasts given low (generally sublethal) doses of UV light. Using 3 distinct assays, we find no sign of a constant repair rate after 4 J/m2 or less, even when the first few hours after irradiation are examined. Thus DNA repair is likely to depend on random collision. The implications of this finding for the structural organisation of repair are discussed.     

PNKP在DNA损伤修复中的作用

多聚核苷酸激酶/磷酸酶(polynucleotide kinase/phosphatase,PNKP)是一种DNA末端修复酶,同时具有激酶和磷酸酶活性,在DNA单链断裂修复途径、碱基切除修复途径以及DNA双链断裂修复中的非同源末端连接途径中发挥着至关重要的作用。近年来,由于一种与PNKP相关的常染色体隐性遗传病——MCSZ综合征的发现,使得人们对PNKP的关注度进一步增加。笔者从与PNKP相互作用的X射线交叉互补修复基因1(X-ray repair cross-complementing group 1,XRCC1)、X射线交叉互补修复基因4(X-ray repair cross-complementing group 4,XRCC4)和毛细血管扩张性共济失调突变基因(ataxia-telangiectasia mutated,ATM)入手,对PNKP在DNA损伤修复中的作用进行概述。     

Repair and mutagenesis survey of 8-hydroxyguanine in bacteria and human cells

8-Hydroxyguanine is one of the major products formed by the reactive oxygen species which are generated in living cells as a consequence of either the normal metabolic pathways or an exogeneous chemical or physical stress. The production of the oxidative damage is described and the different repair pathways of the oxidative lesions are analyzed from bacteria to human cells. Analysis of repair in human cells harboring different deficiencies in the nucleotide excision repair mechanism such as xeroderma pigmentosum cells from different complementation groups and cells from Cockayne's syndrome patients allows us to emphasize the possibility of the intervention of this repair mechanism on the elimination of oxidative damages. Finally, a repair model of oxidative lesions is proposed.     

Current role of mammalian sirtuins in DNA repair

Cellular DNA is constantly challenged by damage-inducing factors derived from exogenous or endogenous sources. Thus, to protect against DNA damage, cells have evolved complex and finely regulated mechanisms collectively known as DNA-damage response (DDR). However, DNA repair in eukaryotes does not occur merely in naked DNA but also within a highly organized and compacted chromatin environment, which ultimately participates in regulating DDR pathways. Thus, remodelling of the chromatin surrounding areas containing damaged DNA is required to allow access to the DNA repair machinery, as well as post-translational modifications in many repair factors to recruit and activate them at the damaged site. Notably, proteins such as sirtuins, which are NAD+-dependent deacetylases, have evolved to modulate multiple repair pathways through deacetylation of some repair factors, influencing chromatin accessibility or indirectly modulating cell cycle and preventing oxidative stress. In this way, the purpose of this review is to summarize the recent knowledge that links sirtuins with DNA repair, with a particular emphasis on the molecular mechanisms associated with coordination and regulation of this vital process.     

A phylogenomic study of DNA repair genes, proteins, and processes

The ability to recognize and repair abnormal DNA structures is common to all forms of life. Studies in a variety of species have identified an incredible diversity of DNA repair pathways. Documenting and characterizing the similarities and differences in repair between species has important value for understanding the origin and evolution of repair pathways as well as for improving our understanding of phenotypes affected by repair (e.g., mutation rates, lifespan, tumorigenesis, survival in extreme environments). Unfortunately, while repair processes have been studied in quite a few species, the ecological and evolutionary diversity of such studies has been limited. Complete genome sequences can provide potential sources of new information about repair in different species. In this paper, we present a global comparative analysis of DNA repair proteins and processes based upon the analysis of available complete genome sequences. We use a new form of analysis that combines genome sequence information and phylogenetic studies into a composite analysis we refer to as phylogenomics. We use this phylogenomic analysis to study the evolution of repair proteins and processes and to predict the repair phenotypes of those species for which we now know the complete genome sequence.