XRCC1 protein is required for DNA single-strand break repair and genetic stability but its biochemical role is unknown. Here, we report that XRCC1 interacts with human polynucleotide kinase in addition to its established interactions with DNA polymerase-beta and DNA ligase III. Moreover, these four proteins are coassociated in multiprotein complexes in human cell extract and together they repair single-strand breaks typical of those induced by reactive oxygen species and ionizing radiation. Strikingly, XRCC1 stimulates the DNA kinase and DNA phosphatase activities of polynucleotide kinase at damaged DNA termini and thereby accelerates the overall repair reaction. These data identify a novel pathway for mammalian single-strand break repair and demonstrate a concerted role for XRCC1 and PNK in the initial step of processing damaged DNA ends.
Polynucleotide kinases catalyze phosphorylation of 5Ј-OH termini of nucleic acids. In a number of biochemical experiments over several decades, evidence for a mammalian polynucleotide kinase (PNK) 1 activities with an acidic pH optimum has mounted (reviewed in Refs. 1-8). We and others have purified such a PNK to near-homogeneity from bovine tissue, which lacks significant 5Ј-phosphorylation activity when assayed with RNA substrates (5, 6, 9). This activity, denoted SNQI-PNK, corresponded to a polypeptide of approximately 60 kDa in our experiments (6). Highly purified SNQI-PNK fractions contain a 3Ј-phosphatase activity (6), originally discovered in the PNK from bacteriophage T4 (10, 11) and also observed in PNKs from rat liver nuclei (2-5, 12). Furthermore, there are reports of mammalian PNK activities with a greater substrate specificity for RNA than DNA (8, 13, 14) 2 and of conservation of yeastlike tRNA ligation (with its requirement for a PNK activity) as a minor pathway in HeLa cells (15).Because of its widespread presence in mammalian cells, the acidic pH optimum PNK is likely to be a key enzyme in DNA metabolism, and its biochemical functions immediately suggest a role in the critical process of DNA repair. One of its enzymatic activities, DNA 3Ј-phosphatase, implies an ability to repair strand breaks terminated by 3Ј-phosphate, a type of DNA damage seen in cells treated with ionizing radiation or hydrogen peroxide (16). Removal of this 3Ј-end blocking lesion allows synthesis by DNA polymerase and joining of nicks by DNA ligase. DNA purified from irradiated thymocytes and irradiated thymus, but not DNA irradiated in vitro, contains strand breaks with 5Ј-OH termini (17, 18). The 5Ј-phosphorylation activity of the SNQI-PNK enzyme suggests a possible model in which 5Ј-OH termini are repaired prior to ligation. 5Ј-OH termini in DNA also occur in ischemia in rat brain (19), after cleavage by nucleases with the appropriate specificity such as DNase II (20), and as intermediates during topoisomerase cleavage (21,22). The highest concentration of 5Ј-DNA termini occurs during DNA replication, and Pohjanpelto and Hölttä (23) proposed that a small fraction of Okazaki fragments contain 5Ј-OH termini; this fraction decreases upon incubation of extracts with ATP at pH 6.0, which was inferred to reflect 5Ј-phosphorylation by a cellular PNK.Despite extensive biochemical studies, to date there are no molecular reagents such as antibodies or cDNAs available for mammalian PNKs, hampering further investigation. We present here the molecular cloning of the PNKP gene, the first gene for a mammalian PNK and the first gene for a DNA-specific kinase from any organism. Concomitantly, the PNKP gene also
Human cDNA clones encoding the major DNA ligase activity in proliferating cells, DNA ligase I, were isolated by two independent methods. In one approach, a human cDNA library was screened by hybridization with oligonucleotides deduced from partial amino acid sequence of purified bovine DNA ligase I. In an alternative approach, a human cDNA library was screened for functional expression of a polypeptide able to complement a cdc9 temperature-sensitive DNA ligase mutant of Saccharomyces cerevisiae. ODS C18 column (100 x 2.1 mm, Brownlee Laboratories) equilibrated with 0.08% trifluoroacetic acid/1% acetonitrile and were eluted with a linear gradient of 1-60% acetonitrile. Partially purified peptides were re-applied to the same column equilibrated with 50 mM ammonium acetate (pH 7.0) and were eluted with a linear gradient of 0-60% acetonitrile. Amino acid sequences of three peptides were determined using a Beckman ABI 477A peptide sequencer.Oligodeoxynucleotide Probes. Single long antisense oligonucleotide probes were designed according to Lathe (11) and synthesized on an Applied Biosystems model 380B DNA synthesizer. Oligonucleotides were 32P-labeled using T4 polynucleotide kinase.Isolation of cDNA Clones by Oligonucleotide Screening. A human Jurkat T-lymphoblast cDNA library in Agtll (obtained from H. Kataoka, Imperial Cancer Research Fund, Clare Hall Laboratories) was screened using 32P-labeled oligonucleotide probes. Hybridization was carried out at 37°C for 18 hr in 6x SSC (0.9 M NaCl/0.09 M sodium citrate, pH 7)/5 x Denhardt's solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin)/0.1% SDS containing sheared, denatured herring sperm DNA (100 ,ug/ml). Filters were washed in 6x SSC/0.1% SDS for 20 min at both 50°C and 55°C.Isolation of cDNA Clones by Complementation in S. cerevisiae. The recipient yeast strain used in transformations was L94-4D (MATa cdc9-7 ura3 trpl), constructed by L.H.J., and its congenic wild-type parent was YNN27 (obtained from
The activation state of Rho is an important determinant of axon growth and regeneration in neurons. Axons can extend neurites on growth inhibitory substrates when Rho is inactivated by C3-ADP-ribosyltransferase (C3). We found by Rho-GTP pull-down assay that inhibitory substrates activate Rho. To inactivate Rho, scrapeloading of C3 was necessary because it does not freely enter cells. To overcome the poor permeability of C3, we made and characterized five new recombinant C3-like chimeric proteins designed to cross the cell membrane by receptor-independent mechanisms. These proteins were constructed by the addition of short transport peptides to the carboxyl-terminal of C3 and tested using a bioassay measuring neurite outgrowth of PC-12 cells plated on growth inhibitory substrates. All five constructs stimulated neurite outgrowth but with different dose-response profiles. Biochemical properties of the chimeric proteins were examined using C3-05, the most effective construct tested. Gel shift assays showed that C3-05 retained the ability to ADP-ribosylate Rho. Western blots and immunocytochemistry were used to verify the presence of C3 inside treated cells. C3-05 was also effective at promoting neurite outgrowth in primary neuronal cultures, as well as causing the disassembly of actin stress fibers and focal adhesions complexes in fibroblasts. These studies demonstrate that the new C3-like proteins are effective in delivering biologically active C3 into different cell types, thereby, inactivating Rho.
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