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Here, we describe the assembly of the nucleotide excision repair (NER) complex in normal and repair-deficient (xeroderma pigmentosum) human cells, employing a novel technique of local UV irradiation combined with fluorescent antibody labeling. The damage recognition complex XPC-hHR23B appears to be essential for the recruitment of all subsequent NER factors in the preincision complex, including transcription repair factor TFIIH. XPA associates relatively late, is required for anchoring of ERCC1-XPF, and may be essential for activation of the endonuclease activity of XPG. These findings identify XPC as the earliest known NER factor in the reaction mechanism, give insight into the order of subsequent NER components, provide evidence for a dual role of XPA, and support a concept of sequential assembly of repair proteins at the site of the damage rather than a preassembled repairosome.

Human Werner Syndrome is characterized by early onset of aging, elevated chromosomal instability, and a high incidence of cancer. Werner protein (WRN) is a member of the recQ gene family, but unlike other members of the recQ family, it contains a unique 335 exonuclease activity. We have reported previously that human Ku heterodimer interacts physically with WRN and functionally stimulates WRN exonuclease activity. Because Ku and DNA-PKcs, the catalytic subunit of DNAdependent protein kinase (DNA-PK), form a complex at DNA ends, we have now explored the possibility of functional modulation of WRN exonuclease activity by DNA-PK. We find that although DNA-PKcs alone does not affect the WRN exonuclease activity, the additional presence of Ku mediates a marked inhibition of it. The inhibition of WRN exonuclease by DNA-PKcs requires the kinase activity of DNA-PKcs. WRN is a target for DNA-PKcs phosphorylation, and this phosphorylation requires the presence of Ku. We also find that treatment of recombinant WRN with a Ser/Thr phosphatase enhances WRN exonuclease and helicase activities and that WRN catalytic activity can be inhibited by rephosphorylation of WRN with DNA-PK. Thus, the level of phosphorylation of WRN appears to regulate its catalytic activities. WRN forms a complex, both in vitro and in vivo, with DNA-PKC. WRN is phosphorylated in vivo after treatment of cells with DNA-damaging agents in a pathway that requires DNA-PKcs. Thus, WRN protein is a target for DNA-PK phosphorylation in vitro and in vivo, and this phosphorylation may be a way of regulating its different catalytic activities, possibly in the repair of DNA dsb. Werner syndrome (WS)1 is a human autosomal recessive disorder characterized by early onset of premature aging characteristics including graying and loss of hair, wrinkling and ulceration of skin, atherosclerosis, osteoporosis, and cataracts. In addition, WS patients exhibit an increased incidence of diabetes mellitus type 2, hypertension, and malignancies (1). The gene (WRN), defects in which are responsible for WS, encodes a 1,432-amino acid protein (WRN) (2) that has both 3Ј35Ј helicase and 3Ј35Ј exonuclease activities (3-7). Although WRN appears to play an important role in DNA metabolism, the precise cellular roles of both the helicase and exonuclease activities of WRN remain to be determined.Cells from patients with WS show premature replicative senesence compared with cells derived from normal individuals (8). The WS cellular phenotype suggests correlations among faulty DNA metabolism, genomic instability, and senescence. WS cells show hypersensitivity to selected DNA-damaging agents including 4-nitroquinoline-1-oxide (4NQO) (9), topoisomerase inhibitors (10), and certain DNA cross-linking agents (11). Compared with normal cells, WS cells also exhibit increased genomic instability including higher levels of DNA deletions, translocations, and chromosomal breaks (12, 13). These studies suggest that WRN plays an important role in DNA metabolism possibly by participating in DNA repair, re...

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The RecQ helicase family comprises a conserved group of proteins implicated in several aspects of DNA metabolism. Three of the family members are defective in heritable diseases characterized by abnormal growth, premature aging, and predisposition to malignancies. These include the WRN and BLM gene products that are defective in Werner and Bloom syndromes, disorders which share many phenotypic and cellular characteristics including spontaneous genomic instability. Here, we report a physical and functional interaction between BLM and WRN. These proteins were coimmunoprecipitated from a nuclear matrix-solubilized fraction, and the purified recombinant proteins were shown to interact directly. Moreover, BLM and WRN colocalized to nuclear foci in three human cell lines. Two regions of WRN that mediate interaction with BLM were identified, and one of these was localized to the exonuclease domain of WRN. Functionally, BLM inhibited the exonuclease activity of WRN. This is the first demonstration of a physical and functional interaction between RecQ helicases. Our observation that RecQ family members interact provides new insights into the complex phenotypic manifestations resulting from the loss of these proteins. Werner syndrome (WS)1 is a hallmark premature aging syndrome associated with increased malignancies and genomic instability (1). The protein defective in WS, Werner syndrome protein (WRN), is an ATP-dependent 3Ј-5Ј-helicase and also has a 3Ј-5Ј-exonuclease activity (2). Recently, a number of protein partners for WRN have been identified. These include proliferating cell nuclear antigen (3), replication protein A (RPA) (4), DNA topoisomerase I (3), the Ku heterodimer (5, 6), DNA polymerase ␦ (7), and p53 (8). Some of these interactions are not only physical but are also functional. Each of these binding proteins is involved in some form of DNA metabolism, such as DNA recombination, replication, and repair, and in the resolution of alternative DNA structures (1, 2). This suggests that WRN plays a role in a number of key DNA metabolic pathways. Bloom syndrome (BS) is a highly cancer-prone disease associated with increased genomic instability and elevated sister chromatid exchanges. The protein defective in this disorder, Bloom syndrome protein (BLM), is a 3Ј-5Ј-helicase (9). BLM binds to RPA, p53, topoisomerase III␣, and RAD51 and is a component of the BRCA1-associated genome surveillance complex (10). This links BLM with proteins implicated in several aspects of DNA metabolism (DNA recombination, replication, and repair).BLM and WRN both belong to the RecQ family of helicases, which are conserved from Escherichia coli to human (11). WRN is unique among the human RecQ helicases in having an exonuclease domain in the N-terminal region of the protein. A number of the RecQ helicases have been purified, and their biochemical properties have been characterized (11). There is considerable interest in the substrate specificities of each of these enzymes and in possible differences between them. However, so far there h...

Summary Rothmund-Thomson syndrome (RTS) is an autosomal recessive hereditary disorder associated with mutation in RECQL4 gene, a member of the human RecQ helicases. The disease is characterized by genomic instability, skeletal abnormalities and predisposition to malignant tumors, especially osteosarcomas. The precise role of RECQL4 in cellular pathways is largely unknown, however recent evidence suggest its involvement in multiple DNA metabolic pathways. This study investigates the roles of RECQL4 in DNA double strand break (DSB) repair. The results show that RECQL4-deficient fibroblasts are moderately sensitive to γ-irradiation and accumulate more γH2AX and 53BP1 foci than control fibroblasts. This is suggestive of defects in efficient repair of DSB’s in the RECQL4 deficient fibroblasts. Real time imaging of live cells using laser confocal microscopy show that RECQL4 is recruited early to laser induced DSBs and remains for a shorter duration than WRN and BLM indicating its distinct role in repair of DSBs. Endogenous RECQL4 also colocalizes with γH2AX at the site of DSBs. The RECQL4 domain responsible for its DNA damage localization has been mapped to the unique N-terminus domain between amino acids 363–492, which shares no homology to recruitment domains of WRN and BLM to the DSBs. Further, the recruitment of RECQL4 to laser induced DNA damage is independent of functional WRN, BLM or ATM proteins. These results suggest distinct cellular dynamics for RECQL4 protein at the site of laser induced DSB and that it might play important roles in efficient repair of DSB’s.

Bactericidal efficacy of gold nanoparticles conjugated with ampicillin, streptomycin and kanamycin were evaluated. Gold nanoparticles (Gnps) were conjugated with the antibiotics during the synthesis of nanoparticles utilizing the combined reducing property of antibiotics and sodium borohydride. The conjugation of nanoparticles was confirmed by dynamic light scattering (DLS) and electron microscopic (EM) studies. Such Gnps conjugated antibiotics showed greater bactericidal activity in standard agar well diffusion assay. The minimal inhibitory concentration (MIC) values of all the three antibiotics along with their Gnps conjugated forms were determined in three bacterial strains,Escherichia coli DH5α,Micrococcus luteusandStaphylococcus aureus. Among them, streptomycin and kanamycin showed significant reduction in MIC values in their Gnps conjugated form whereas; Gnps conjugated ampicillin showed slight decrement in the MIC value compared to its free form. On the other hand, all of them showed more heat stability in their Gnps conjugated forms. Thus, our findings indicated that Gnps conjugated antibiotics are more efficient and might have significant therapeutic implications.