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Unlike most DNA viruses, poxviruses replicate in the cytoplasm of host cells. They encode enzymes needed for genome replication and transcription, including their own thymidine and thymidylate kinases. Some herpes viruses encode only 1 enzyme catalyzing both reactions, a peculiarity used for prodrug activation to obtain maximum specificity. We have solved the crystal structures of vaccinia virus thymidylate kinase bound to TDP or brivudin monophosphate. Although the viral and human enzymes have similar sequences (42% identity), they differ in their homodimeric association and active-site geometry. The vaccinia TMP kinase dimer arrangement is orthogonal and not antiparallel as in human enzyme. This different monomer orientation is related to the presence of a canal connecting the edge of the dimer interface to the TMP base binding pocket. Consequently, the pox enzyme accommodates nucleotides with bulkier bases, like brivudin monophosphate and dGMP; these are efficiently phosphorylated and stabilize the enzyme. The brivudin monophosphate-bound structure explains the structural basis for this specificity, opening the way to the rational development of specific antipox agents that may also be suitable for poxvirus TMP kinase gene-based chemotherapy of cancer. BVdU | dihalovinyl-dU | dimerization mode | TMP kinase | vaccinia virus

Trimethoprim-sulfamethoxazole (SXT)-resistant Staphylococcus aureus thymidine-dependent small-colony variants (TD-SCVs) are frequently isolated from the airways of cystic fibrosis (CF) patients, often in combination with isogenic normal strains if patients were treated with SXT for extended periods. As SXT inhibits the synthesis of tetrahydrofolic acid, which acts as a cofactor for thymidylate synthase (thyA), the survival of TD-SCVs depends exclusively on the availability of external thymidine. Since the underlying mechanism for thymidine dependency is unknown, we investigated if alterations in the thyA nucleotide sequences were responsible for this phenomenon. Sequence analysis of several clinical TD-SCVs and their isogenic normal strains with reference to previously published S. aureus thyA nucleotide sequences was performed. Three clinical TD-SCVs were complemented by transforming TD-SCVs with the vector pCX19 expressing ThyA from S. aureus 8325-4. Transcriptional analysis of metabolic and virulence genes and regulators (agr, hla, spa, citB, thyA, and nupC) was performed by quantitative reverse transcription-PCR. The previously published sequences ofthyA and two normal clinical strains were highly conserved, while thyA of four normal strains and four SCVs had nonsynonymous point mutations. In 8/10 SCVs, deletions occurred, resulting in stop codons which were Iocated in 4/10 SCVs close to or within the active site of the protein (dUMP binding). Complementation of TD-SCVs with thyA almost fully reversed the phenotype, growth characteristics, and transcription patterns. In conclusion, we demonstrated that mutations of the thyA gene were responsible for the phenotype of TD-SCVs. Complementation of TD-SCVs with thyA revealed that a functional ThyA protein is necessary and suflicient to change the SCV phenotype and behavior back to normal.

ABSTRACT Given the increasing commercial and clinical relevance of probiotic cultures, improving their stress tolerance profile and ability to overcome the physiochemical defences of the host is an important biological [...]

We examined the mechanism by which recombination between imperfectly matched sequences (homeologous recombination) is suppressed in mammalian chromosomes. DNA substrates were constructed, each containing a thymidine kinase (tk) gene disrupted by insertion of an XhoI linker and referred to as a 'recipient' gene. Each substrate also contained one of several 'donor' tk sequences that could potentially correct the recipient gene via recombination. Each donor sequence either was perfectly homologous to the recipient gene or contained homeologons sequence sharing only 80% identity with the recipient gene. Mouse L [tk.sup.-] fibroblasts were stably transfected with the various substrates and [tk.sup.+] segregants produced via intrachromosomal recombination were recovered. We observed exclusion of homeologous sequence from gene conversion tracts when homeologous sequence was positioned adjacent to homologous sequence in the donor but not when homeologous sequence was surrounded by homology in the donor. Our results support a model in which homeologous recombination in mammalian chromosomes is suppressed by a nondestructive dismantling of mismatched heteroduplex DNA (hDNA) intermediates. We suggest that mammalian cells do not dismantle mismatched hDNA by responding to mismatches in hDNA per se but rather rejection of mismatched hDNA appears to be driven by a requirement for localized homology for resolution of recombination.

Research has been conducted on the microsatellite DNA polymerase-mediated beta mutations. The polymerase error frequencies at the microsatellite sequences have been investigated in elucidating the microsatellite mutation's molecular basis and the details are reported.

Research has been conducted on the encoded by herpes simplex virus type 1 thymidine kinase. Results indicate that the single A167Y mutation can convert this thymidine kinase into the exclusive purine (guanine) nucleoside analogue kinase.

Participatory sequences in the herpes simplex virus thymidine kinase (HSV tk) recoding are determined in a herpesvirus mutant. The findings demonstrate a novel mechanism that relies on the capacity of the G-rich sequences to generate unique inter- or intramolecular structures. Specifically, the sole influence of G-strings to activate a net +1 recoding in cultured cells is determined. On the other hand, mediation of recoding by the G-string structure inside the ribosome is proposed to proceed either by the Hoogsteen or non-Watson-Crick interactions.

The role of the thymine (T) tract in the structure and stability of model telomeric sequences was analyzed. It was shown that the tetrameric structure is influenced by the T residue flanking either side of the G-cluster. The formation of dimer or tetramer depends on both the length of the T tract and the presence of Na+ or K+. In K+, the predominant structure formed is the parallel four-stranded tetramer. At least two T residues are required to allow formation of folded-back structures.

The studies of comparative mutagenesis of [gamma]- or X-ray-induced tandem DNA lesions G[8,5-Me]T and T[5-Me,8]G intrastrand cross-links in simian (COS-7) and human embyronic (293T) kidney cells are presented. The results from in vitro bypass study of G[8,5-Me]T suggested that human polymerase [eta] (hpol [eta]) might be involved in the correct mutagenic bypass to incorporate dNMP opposite each cross-linked base.

The design, preparation, and properties of two key building blocks of a size-expanded genetic system is described. The nucleoside analogues of the natural nucleosides dA and dT are reported in which the fusion of a benzo ring increases their size.