The paradigm for repair of oxidized base lesions in genomes via the base excision repair (BER) pathway is based on studies in Escherichia coli, in which AP endonuclease (APE) removes all 3' blocking groups (including 3' phosphate) generated by DNA glycosylase/AP lyases after base excision. The recently discovered mammalian DNA glycosylase/AP lyases, NEIL1 and NEIL2, unlike the previously characterized OGG1 and NTH1, generate DNA strand breaks with 3' phosphate termini. Here we show that in mammalian cells, removal of the 3' phosphate is dependent on polynucleotide kinase (PNK), and not APE. NEIL1 stably interacts with other BER proteins, DNA polymerase beta (pol beta) and DNA ligase IIIalpha. The complex of NEIL1, pol beta, and DNA ligase IIIalpha together with PNK suggests coordination of NEIL1-initiated repair. That NEIL1/PNK could also repair the products of other DNA glycosylases suggests a broad role for this APE-independent BER pathway in mammals.

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.

Mammalian polynucleotide kinase (PNK) is a key component of both the base excision repair (BER) and nonhomologous end-joining (NHEJ) DNA repair pathways. PNK acts as a 5'-kinase/3'-phosphatase to create 5'-phosphate/3'-hydroxyl termini, which are a necessary prerequisite for ligation during repair. PNK is recruited to repair complexes through interactions between its N-terminal FHA domain and phosphorylated components of either pathway. Here, we describe the crystal structure of intact mammalian PNK and a structure of the PNK FHA bound to a cognate phosphopeptide. The kinase domain has a broad substrate binding pocket, which preferentially recognizes double-stranded substrates with recessed 5' termini. In contrast, the phosphatase domain efficiently dephosphorylates single-stranded 3'-phospho termini as well as double-stranded substrates. The FHA domain is linked to the kinase/phosphatase catalytic domain by a flexible tether, and it exhibits a mode of target selection based on electrostatic complementarity between the binding surface and the phosphothreonine peptide.

Human polydeoxyribonucleotide kinase is an enzyme that has the capacity to phosphorylate DNA at 5-hydroxyl termini and dephosphorylate 3-phosphate termini and, therefore, can be considered a putative DNA repair enzyme. The enzyme was purified from HeLa cells. Amino acid sequence was obtained for several tryptic fragments by mass spectrometry. The sequences were matched through the dbEST data base with an incomplete human cDNA clone, which was used as a probe to retrieve the 5-end of the cDNA sequence from a separate cDNA library. The complete cDNA, which codes for a 521-amino acid protein (57.1 kDa), was expressed in Escherichia coli, and the recombinant protein was shown to possess the kinase and phosphatase activities. Comparison with other sequenced proteins identified a P-loop motif, indicative of an ATP-binding domain, and a second motif associated with several different phosphatases. There is reasonable sequence similarity to putative open reading frames in the genomes of Caenorhabditis elegans and Schizosaccharomyces pombe, but similarity to bacteriophage T4 polynucleotide kinase is limited to the kinase and phosphatase domains noted above. Northern hybridization revealed a major transcript of approximately 2.3 kilobases and a minor transcript of approximately 7 kilobases. Pancreas, heart, and kidney appear to have higher levels of mRNA than brain, lung, or liver. Confocal microscopy of human A549 cells indicated that the kinase resides predominantly in the nucleus. The gene encoding the enzyme was mapped to chromosome band 19q13.4.Transient DNA strand breaks and short gaps are frequently observed in cellular DNA. Many arise during regular cellular activity such as DNA replication, recombination, or differentiation. Others occur as a consequence of exposure to endogenous or exogenous DNA damaging agents. Repair of these strand interruptions is usually mediated by DNA ligases and polymerases. Both of these classes of enzymes require 3Ј-hydroxyl DNA termini, and the DNA ligases also require 5Ј-phosphate termini. However, the termini generated by nucleases, such as DNase II, and many produced by ionizing radiation bear 3Ј-phosphate and 5Ј-hydroxyl groups (1-4), and therefore must be processed before they can be acted upon by DNA ligases or polymerases.One enzyme that possesses the capacity to both phosphorylate 5Ј-hydroxyl termini and dephosphorylate 3Ј-phosphate termini is polynucleotide kinase (PNK).1 The PNK from T4 phage has found widespread application in molecular biology, especially for radiolabeling DNA and oligonucleotides (5). It can act on DNA and RNA and even phosphorylate nucleoside 3Ј-monophosphates. However, the main cellular function of the T4 enzyme is not to repair DNA, but rather to counter the action of a phage endoribonuclease that cleaves tRNA (6). Eukaryotic PNKs fall into two categories depending on whether their preferred substrate is DNA or RNA (7). While both can phosphorylate 5Ј-termini, only the former have an associated 3Ј-phosphatase activity (8 -12).Mammalian DNA kinases have ...

Termination of wound healing requires a fine balance between collagen deposition and its hydrolysis. To dissect the underlying control mechanisms for this process, we established a keratinocyte/fibroblast co-culture system and subsequently demonstrated more than a 10-fold increase in collagenase expression in fibroblasts co-cultured with keratinocytes relative to that of control cells. This finding was further confirmed in fibroblasts grown in a keratinocyte/fibroblast collagen-GAG gel. The efficacy of keratinocyte-derived collagenase stimulatory factors on collagenase activity was evaluated, and the results showed that only conditioned medium derived from fibroblasts co-cultured with keratinocytes was able to break down markedly type I collagen to its one-quarter and three-quarter fragments of both alpha (alpha1 and alpha2) and beta (beta1.1 and beta1.2) chains. The results of a dose-response experiment showed that keratinocyte-conditioned medium (KCM) stimulates the expression of collagenase mRNA by dermal fibroblasts in a concentration-dependent fashion. In a similar experiment, the results of a time-response experiment revealed that KCM treatment increases the expression of collagenase mRNA in dermal fibroblasts as early as 6 h and reaches its maximum level within 24-48 h. Considering that this keratinocyte-releasable factor has a potent collagenase stimulatory effect on fibroblasts, which favors the resolution of accumulated type I and type III collagen found in fibrotic tissue, we referred to this protein as a keratinocyte-derived anti-fibrogenic factor (KDAF). In a series of chromatography experiments and a direct trypsin digestion of the proteins and subsequent peptide mapping, a keratinocyte-derived collagenase-stimulating factor turned out to be a releasable form of stratifin, also known as 14-3-3 sigma protein. To validate this finding, stratifin cDNA was cloned into a pGEX-6P-1 expressing vector and more than 50 mg of recombinant stratifin was generated and used to treat fibroblasts with various concentrations for 24 h. The results of northern analysis showed a remarkable dose-response increase in the expression of collagenase mRNA in stratifin-treated fibroblasts relative to that of the control. This finding was consistent with that obtained from collagenase activity assay. In conclusion, we identified a keratinocyte-releasable form of stratifin in KCM that mimics the collagenase stimulatory effect of KCM for dermal fibroblasts. This finding suggests that stratifin is likely to be, at least, one of the KDAFs found in KCM.