Characterization of the adenylation site in the RNA 3'-terminal phosphate cyclase from Escherichia coli. (33/1149)

RNA 3'-terminal phosphate cyclases are a family of evolutionarily conserved enzymes that catalyze ATP-dependent conversion of the 3'-phosphate to the 2',3'-cyclic phosphodiester at the end of RNA. The precise function of cyclases is not known, but they may be responsible for generating or regenerating cyclic phosphate RNA ends required by eukaryotic and prokaryotic RNA ligases. Previous work carried out with human and Escherichia coli enzymes demonstrated that the initial step of the cyclization reaction involves adenylation of the protein. The AMP group is then transferred to the 3'-phosphate in RNA, yielding an RNA-N(3')pp(5')A (N is any nucleoside) intermediate, which finally undergoes cyclization. In this work, by using different protease digestions and mass spectrometry, we assign the site of adenylation in the E. coli cyclase to His-309. This histidine is conserved in all members of the class I subfamily of cyclases identified by phylogenetic analysis. Replacement of His-309 with asparagine or alanine abrogates both enzyme-adenylate formation and cyclization of the 3'-terminal phosphate in a model RNA substrate. The cyclase is the only known protein undergoing adenylation on a histidine residue. Sequences flanking the adenylated histidine in cyclases do not resemble those found in other proteins modified by nucleotidylation.  (+info)

An exonic splicing silencer in the testes-specific DNA ligase III beta exon. (34/1149)

Alternative pre-mRNA splicing of two terminal exons (alpha and beta) regulates the expression of the human DNA ligase III gene. In most tissues, the alpha exon is expressed. In testes and during spermatogenesis, the beta exon is used instead. The alpha exon encodes the interaction domain with a scaffold DNA repair protein, XRCC1, while the beta exon-encoded C-terminal does not. Sequence elements regulating the alternative splicing pattern were mapped by in vitro splicing assays in HeLa nuclear extracts. Deletion of a region beginning in the beta exon and extending into the downstream intron derepressed splicing to the beta exon. Two silencing elements were found within this 101 nt region: a 16 nt exonic splicing silencer immediately upstream of the beta exon polyadenylation signal and a 45 nt intronic splicing silencer. The exonic splicing silencer inhibited splicing, even when the poly-adenylation signal was deleted or replaced by a 5' splice site. This element also enhanced polyadenylation under conditions unfavourable to splicing. The splicing silencer partially inhibited assembly of spliceo-somal complexes and functioned in an adenoviral pre-mRNA context. Silencing of splicing by the element was associated with cross-linking of a 37 kDa protein to the RNA substrate. The element exerts opposite functions in splicing and polyadenylation.  (+info)

A phylogenomic study of DNA repair genes, proteins, and processes. (35/1149)

The ability to recognize and repair abnormal DNA structures is common to all forms of life. Studies in a variety of species have identified an incredible diversity of DNA repair pathways. Documenting and characterizing the similarities and differences in repair between species has important value for understanding the origin and evolution of repair pathways as well as for improving our understanding of phenotypes affected by repair (e.g., mutation rates, lifespan, tumorigenesis, survival in extreme environments). Unfortunately, while repair processes have been studied in quite a few species, the ecological and evolutionary diversity of such studies has been limited. Complete genome sequences can provide potential sources of new information about repair in different species. In this paper, we present a global comparative analysis of DNA repair proteins and processes based upon the analysis of available complete genome sequences. We use a new form of analysis that combines genome sequence information and phylogenetic studies into a composite analysis we refer to as phylogenomics. We use this phylogenomic analysis to study the evolution of repair proteins and processes and to predict the repair phenotypes of those species for which we now know the complete genome sequence.  (+info)

The nonhomologous DNA end joining pathway is important for chromosome stability in primary fibroblasts. (36/1149)

There are two types of chromosome instability, structural and numerical, and these are important in cancer. Many structural abnormalities are likely to involve double-strand DNA (dsDNA) breaks. Nonhomologous DNA end joining (NHEJ) and homologous recombination are the major pathways for repairing dsDNA breaks. NHEJ is the primary pathway for repairing dsDNA breaks throughout the G0, G1 and early S phases of the cell cycle [1]. Ku86 and DNA ligase IV are two major proteins in the NHEJ pathway. We examined primary dermal fibroblasts from mice (wild type, Ku86(+/-), Ku86(-/-), and DNA ligase IV(+/-)) for chromosome breaks. Fibroblasts from Ku86(+/-) or DNA ligase IV(+/-) mice have elevated frequencies of chromosome breaks compared with those from wild-type mice. Fibroblasts from Ku86(-/-) mice have even higher levels of chromosome breaks. Primary pre-B cells from the same animals did not show significant accumulation of chromosome breaks. Rather the pre-B cells showed increased cell death. These studies demonstrate that chromosome breaks arise frequently and that NHEJ is required to repair this constant spontaneous damage.  (+info)

A cell cycle-specific requirement for the XRCC1 BRCT II domain during mammalian DNA strand break repair. (37/1149)

XRCC1 protein is essential for viability in mammals and is required for efficient DNA single-strand break repair and genetic stability following DNA base damage. We report here that XRCC1-dependent strand break repair in G(1) phase of the cell cycle is abolished by mutations created within the XRCC1 BRCT domain that interact with DNA ligase III. In contrast, XRCC1-dependent DNA strand break repair in S phase is largely unaffected by these mutations. These data describe a cell cycle-specific role for a BRCT domain, and we conclude that the XRCC1-DNA ligase III complex is required for DNA strand break repair in G(1) phase of the cell cycle but is dispensable for this process in S phase. The S-phase DNA repair process can remove both strand breaks induced in S phase and those that persist from G(1) and can in part compensate for lack of repair in G(1). This process correlates with the appearance of XRCC1 nuclear foci that colocalize with Rad51 and may thus function in concert with homologous recombination.  (+info)

Impact of menstrual cycle on the diagnostic performance of LCR, TMA, and PCE for detection of Chlamydia trachomatis in home obtained and mailed vaginal flush and urine samples. (38/1149)

OBJECTIVES: To assess the impact of the menstrual cycle on the diagnostic performance of various assays for detection of Chlamydia trachomatis in home obtained and mailed vaginal flush and urine specimens. METHODS: A ligase chain reaction assay (LCR; Abbott Laboratories), a transcription mediated amplification assay (TMA; Gen-Probe), and an enzyme amplified immunoassay (PCE; Dako Diagnostics) were evaluated for their validity in detecting C trachomatis in vaginal flush, first void urine, and midstream urine specimens obtained by female high school students at home and mailed directly to the diagnostic laboratory. RESULTS: C trachomatis was detected in 45 of 889 females (5.1%). The vaginal flush material was positive by TMA and LCR in 84% and 82% of the chlamydia positive females, respectively. First void urine was positive by TMA in 73% and by LCR in 49% of the cases. Midstream urine was positive by TMA and LCR in 69% and 42% of the females, respectively. On a pool of first void and midstream urine, PCE detected 49% of the chlamydia positive females. The overall prevalence of C trachomatis increased with increasing time after the last menstrual bleeding. In urine samples, but not vaginal flush specimens, obtained 3 weeks after the last menstrual bleeding, the sensitivities of TMA, LCR, and PCE decreased markedly suggesting that inhibitors to the assays are excreted in the urine but not in vaginal secretions at this time. CONCLUSION: Vaginal flush samples are superior to urines for detection of chlamydia infections in females. In screening of young asymptomatic females, samples should be obtained in the latter part of the menstrual cycle.  (+info)

Characterization of in vivo developmental chromosome fragmentation intermediates in E. crassus. (39/1149)

Ligation-mediated PCR was used to characterize intermediates in the fragmentation/de novo telomere addition process that occurs during sexual reproduction in the ciliate E. crassus. Fragmentation generates ends with 6-base, 3' overhangs that have 5'-phosphate and 3'-hydroxyl groups. These intermediates are detected only during the period of chromosome fragmentation. Fragmentation always occurs at a precise distance from a conserved sequence, the E-Cbs, indicating that it is a key cis-acting element in the process. The results also serve to identify the natural substrate for de novo telomere addition and indicate that telomerase recognizes, and compensates for, partial telomeric repeats at the ends of fragmentation intermediates. Similarities of the Euplotes fragmentation/telomere addition process to the movement of some non-long terminal repeat retrotransposons are discussed.  (+info)

Topical treatment with liposomes containing T4 endonuclease V protects human skin in vivo from ultraviolet-induced upregulation of interleukin-10 and tumor necrosis factor-alpha. (40/1149)

Exposing human skin to ultraviolet radiation causes DNA damage, sunburn, immune alterations, and eventually, skin cancer. We wished to determine whether liposomes containing a DNA repair enzyme could prevent any of the acute effects of irradiation when applied after ultraviolet exposure. Fifteen human patients with a prior history of skin cancer were exposed to two minimal erythema doses of ultraviolet radiation on their buttock skin. Liposomes containing T4 endonuclease V or heat-inactivated enzyme were applied immediately and at 2, 4, and 5 h after ultraviolet irradiation. Transmission electron microscopy after anti-T4 endonuclease V-staining and immunogold labeling on biopsies taken at 6 h after ultraviolet exposure revealed that the enzyme was present within cells in the skin. Immunohistochemical DNA damage studies suggested a trend toward improved DNA repair at the active T4 endonuclease V liposome-treated test sites. Although the active T4 endonuclease V liposomes did not significantly affect the ultraviolet-induced erythema response and microscopic sunburn cell formation, they nearly completely prevented ultraviolet-induced upregulation of interleukin-10 and tumor necrosis factor-alpha RNA message and of interleukin-10 protein. These studies demonstrate that liposomes can be used for topical intracellular delivery of small proteins to human skin and suggest that liposomes containing DNA repair enzymes may provide a new avenue for photoprotection against some forms of ultraviolet-induced skin damage.  (+info)