Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases

Xeroderma pigmentosum is a monogenic disease characterized by hypersensitivity to ultraviolet light. The cells of xeroderma pigmentosum patients are defective in nucleotide excision repair, limiting their capacity to eliminate ultraviolet-induced DNA damage, and resulting in a strong predisposition to develop skin cancers. The use of rare cutting DNA endonucleases-such as homing endonucleases, also known as meganucleases-constitutes one possible strategy for repairing DNA lesions. Homing endonucleases have emerged as highly specific molecular scalpels that recognize and cleave DNA sites, promoting efficient homologous gene targeting through double-strand-break-induced homologous recombination. Here we describe two engineered heterodimeric derivatives of the homing endonuclease I-CreI, produced by a semi-rational approach. These two molecules-Amel3-Amel4 and Ini3-Ini4-cleave DNA from the human XPC gene (xeroderma pigmentosum group C), in vitro and in vivo. Crystal structures of the I-CreI variants complexed with intact and cleaved XPC target DNA suggest that the mechanism of DNA recognition and cleavage by the engineered homing endonucleases is similar to that of the wild-type I-CreI. Furthermore, these derivatives induced high levels of specific gene targeting in mammalian cells while displaying no obvious genotoxicity. Thus, homing endonucleases can be designed to recognize and cleave the DNA sequences of specific genes, opening up new possibilities for genome engineering and gene therapy in xeroderma pigmentosum patients whose illness can be treated ex vivo.     

Isolation of UV-resistant revertants from a xeroderma pigmentosum complementation group A cell line

Xeroderma pigmentosum (XP) is an autosomal recessive disease. Cells cultured from XP patients are hypersensitive to the lethal effects of UV light. Most XP cells are defective in an early stage in DNA repair of UV light-induced damage. The nature of the genetic defect of the XP syndrome has not been defined. To address this problem, we attempted to isolate UV-resistant cells from a cell line derived from an XP complementation group A (XPA) patient. By using a selection scheme capable of detecting one UV-resistant cell in a population of 10(8) cells, several UV-resistant clones were isolated at frequencies between 1 X 10(-7) and 2 X 10(-8). Here we describe the isolation and initial characterization of these phenotypic revertants.     

The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta.

Xeroderma pigmentosum variant (XP-V) is an inherited disorder which is associated with increased incidence of sunlight-induced skin cancers. Unlike other xeroderma pigmentosum cells (belonging to groups XP-A to XP-G), XP-V cells carry out normal nucleotide-excision repair processes but are defective in their replication of ultraviolet-damaged DNA. It has been suspected for some time that the XPV gene encodes a protein that is involved in trans-lesion DNA synthesis, but the gene product has never been isolated. Using an improved cell-free assay for trans-lesion DNA synthesis, we have recently isolated a DNA polymerase from HeLa cells that continues replication on damaged DNA by bypassing ultraviolet-induced thymine dimers in XP-V cell extracts. Here we show that this polymerase is a human homologue of the yeast Rad30 protein, recently identified as DNA polymerase eta. This polymerase and yeast Rad30 are members of a family of damage-bypass replication proteins which comprises the Escherichia coli proteins UmuC and DinB and the yeast Rev1 protein. We found that all XP-V cells examined carry mutations in their DNA polymerase eta gene. Recombinant human DNA polymerase eta corrects the inability of XP-V cell extracts to carry out DNA replication by bypassing thymine dimers on damaged DNA. Together, these results indicate that DNA polymerase eta could be the XPV gene product.     

Yeast RAD14 and human xeroderma pigmentosum group A DNA-repair genes encode homologous proteins.

Xeroderma pigmentosum (XP), a human autosomal recessive disorder, is characterized by extreme sensitivity to sunlight and high incidence of skin cancers. XP cells are defective in the incision step of excision repair of DNA damaged by ultraviolet light. Cell fusion studies have defined seven XP complementation groups, XP-A to XP-G. Similar genetic complexity of excision repair is observed in the yeast Saccharomyces cerevisiae. Mutations in any one of five yeast genes, RAD1, RAD2, RAD3, RAD4, and RAD10, cause a total defect in incision and an extreme sensitivity to ultraviolet light. Here we report the characterization of the yeast RAD14 gene. The available rad14 point mutant is only moderately ultraviolet-sensitive, and it performs a substantial amount of incision of damaged DNA. Our studies with the rad14 deletion (delta) mutation indicate an absolute requirement of RAD14 in incision. RAD14 encodes a highly hydrophilic protein of 247 amino acids containing zinc-finger motifs, and it is similar to the protein encoded by the human XPAC gene that complements XP group A cell lines.     

Analysis of a human DNA excision repair gene involved in group A xeroderma pigmentosum and containing a zinc-finger domain

Xeroderma pigmentosum (XP) is an autosomal recessive disease, characterized by a high incidence of sunlight-induced skin cancer. Cells from people with this condition are hypersensitive to ultraviolet because of a defect in DNA repair. There are nine genetic complementation groups of XP, groups A-H and a variant. We have cloned the mouse DNA repair gene that complements the defect of group A, the XPAC gene. Here we report molecular cloning of human and mouse XPAC complementary DNAs. Expression of XPAC cDNA confers ultraviolet-resistance on several group A cell lines, but not on lines of other XP groups. Almost all group A lines tested showed abnormality or absence of XPAC messenger RNAs. These results indicate that a defective XPAC gene causes group A XP. The human and mouse XPAC genes are located on chromosome 9q34.1 and chromosome 4C2, respectively. Human XPAC cDNA encodes a protein of 273 amino acids with a zinc-finger motif.     

A low fraction of nitrogen in molecular form in a dark cloud

Nitrogen is the fifth most abundant element in the Universe. In the interstellar medium, it has been thought to be mostly molecular (N2). However, N2 has no observable rotational or vibrational transitions, so its abundance in the interstellar medium remains poorly known. In comets, the N2 abundance is very low, while the elemental nitrogen abundance is deficient with respect to the solar value. Moreover, large nitrogen isotopic anomalies are observed in meteorites and interstellar dust particles. Here we report the N2H+ (and by inference the N2) abundance inside a cold dark molecular cloud. We find that only a small fraction of nitrogen in the gas phase is molecular, with most of it being atomic. Because the compositions of comets probably reflect those of dark clouds, this result explains the low N2 abundance in comets. We argue that the elemental nitrogen abundance deficiency in comets can be understood if the atomic oxygen abundance is lower than predicted by present chemical models. Furthermore, the lack of molecular nitrogen in molecular clouds explains the nitrogen anomalies in meteorites and interstellar dust particles, as nitrogen fractionation is enhanced if gaseous nitrogen is atomic.     

新媒体时代绘画的叙事方式

科技飞速发展为人们带来很多便利,数字媒体技术的成熟也为艺术家的创作开拓了更广阔的空间。本文结合时下绘画创作,阐述新媒体时代绘事语言的嬗变。     

若尔当标准形问题新探

给出了“每一个n级复数矩阵A都与一个若尔当矩阵相似,且这个若尔当形矩阵除去其中若尔当块的排列次序外是被矩阵A唯一决定的.”这个定理的一个新证法.