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Escherichia coli nucleoside diphosphate (NDP) kinase mutants have an increased frequency of spontaneous mutation, possibly due to uracil misincorporation into DNA. Here we show that NDP kinase mutants are dependent on translesion DNA synthesis, often a mutagenic form of DNA synthesis, to prevent mutagenesis.
Loss of nucleoside diphosphate kinase (Ndk) function in Escherichia coli results in an increased frequency of spontaneous mutation and an imbalance in dNTP pool levels. It is presumed that the imbalance in dNTP pool levels is responsible for the mutator phenotype of an E. coli ndk mutant. A human homologue of Ndk and potential suppressor of tumor metastasis, nm23-H2, can complement the mutagenic phenotype of an E. coli ndk mutant. Here, we show that the antimutagenic property of nm23-H2 in E. coli is independent of dNTP pool levels, indicating that dNTP pool imbalance is not responsible for the mutator phenotype associated with the loss of ndk function. We have identified multiple genetic interactions between ndk and genes involved in the metabolism of dUTP, a potentially mutagenic precursor of thymidine biosynthesis. We show that loss of ndk function is synergistic with a dut-1 mutation and synthetically lethal with the loss of thymidine kinase function. Our results suggest that Ndk prevents the accumulation of dUTP in vivo. Based on these results and biochemical studies of Ndk, we propose that the mutagenic phenotype of an ndk mutant is caused by excess misincorporation of uracil in place of thymidine combined with a defect in the uracil base excision pathway.
We present a computational model of mitochondrial deoxynucleotide metabolism and mitochondrial DNA (mtDNA) synthesis. The model includes the transport of deoxynucleosides and deoxynucleotides into the mitochondrial matrix space, as well as their phosphorylation and polymerization into mtDNA. Different simulated cell types (cancer, rapidly dividing, slowly dividing, and postmitotic cells) are represented in this model by different cytoplasmic deoxynucleotide concentrations. We calculated the changes in deoxynucleotide concentrations within the mitochondrion during the course of a mtDNA replication event and the time required for mtDNA replication in the different cell types. On the basis of the model, we define three steady states of mitochondrial deoxynucleotide metabolism: the phosphorylating state (the net import of deoxynucleosides and export of phosphorylated deoxynucleotides), the desphosphorylating state (the reverse of the phosphorylating state), and the efficient state (the net import of both deoxynucleosides and deoxynucleotides). We present five testable hypotheses based on this simulation. First, the deoxynucleotide pools within a mitochondrion are sufficient to support only a small fraction of even a single mtDNA replication event. Second, the mtDNA replication time in postmitotic cells is much longer than that in rapidly dividing cells. Third, mitochondria in dividing cells are net sinks of cytoplasmic deoxynucleotides, while mitochondria in postmitotic cells are net sources. Fourth, the deoxynucleotide carrier exerts the most control over the mtDNA replication rate in rapidly dividing cells, but in postmitotic cells, the NDPK and TK2 enzymes have the most control. Fifth, following from the previous hypothesis, rapidly dividing cells derive almost all of their mtDNA precursors from the cytoplasmic deoxynucleotides, not from phosphorylation within the mitochondrion.
We have identified a new pyrimidine-tract binding factor, PUF, that is required, together with U2AF, for efficient reconstitution of RNA splicing in vitro. The activity has been purified and consists of two proteins, PUF60 and the previously described splicing factor p54. p54 and PUF60 form a stable complex in vitro when cotranslated in a reaction mixture. PUF activity, in conjunction with U2AF, facilitates the association of U2 snRNP with the pre-mRNA. This reaction is dependent upon the presence of the large subunit of U2AF, U2AF65, but not the small subunit U2AF35. PUF60 is homologous to both U2AF65 and the yeast splicing factor Mud2p. The C-terminal domain of PUF60, the PUMP domain, is distantly related to the RNA-recognition motif domain, and is probably important in protein-protein interactions.
BACKGROUND - The two major types of noninvasive breast carcinoma, ductal carcinoma in situ (DCIS) and lobular carcinoma in situ (LCIS), are quite different in their histopathologic appearance and clinical implications. LCIS is only a marker of an increased risk of later development of invasive carcinoma, whereas most DCIS lesions are at least nonobligate precursors of invasive carcinoma. DCIS is a heterogeneous group of lesions composed of several distinct subtypes, with only the comedo subtype having immediate malignant potential. The authors' purpose was to analyze noninvasive carcinomas for the presence of a gene product (nm23) indicative of a favorable prognosis in invasive carcinomas to determine differences (1) among the different types of CIS and (2) in CIS with and without an accompanying invasive component.
METHODS - Immunohistochemical methods were used to detect nm23 gene product in archival material from two groups of patients: Group 1 consisted of 54 cases of purely noninvasive carcinoma, and Group 2 consisted of 55 examples of noninvasive carcinoma associated with an invasive component.
RESULTS - Among the cases of CIS with no invasion, LCIS and comedo DCIS expressed more nm23 than noncomedo DCIS (P < or = 0.03). There were no differences among these CIS subtypes in the group with invasion. Comparing subtypes of CIS in the groups with or without invasion, only comedo DCIS was significantly different, with greater expression in the CIS group with no invasion compared with comedo DCIS associated with an invasive component (P = 0.04).
CONCLUSIONS - These results support the special nature of LCIS and the heterogeneous nature of DCIS. The in situ component attending an invasive component may be different from anatomically similar lesion without associated invasion. The absence of nm23 in comedo DCIS may be indicative of invasive capacity.