DNMT1 but not its interaction with the replication machinery is required for maintenance of DNA methylation in human cells.

DNA methylation plays a central role in the epigenetic regulation of gene expression in vertebrates. Genetic and biochemical data indicated that DNA methyltransferase 1 (Dnmt1) is indispensable for the maintenance of DNA methylation patterns in mice, but targeting of the DNMT1 locus in human HCT116 tumor cells had only minor effects on genomic methylation and cell viability. In this study, we identified an alternative splicing in these cells that bypasses the disrupting selective marker and results in a catalytically active DNMT1 protein lacking the proliferating cell nuclear antigen-binding domain required for association with the replication machinery. Using a mechanism-based trapping assay, we show that this truncated DNMT1 protein displays only twofold reduced postreplicative DNA methylation maintenance activity in vivo. RNA interference-mediated knockdown of this truncated DNMT1 results in global genomic hypomethylation and cell death. These results indicate that DNMT1 is essential in mouse and human cells, but direct coupling of the replication of genetic and epigenetic information is not strictly required.

Hydrogen peroxide-producing lactobacilli inhibit gonococci in vitro but not during experimental genital tract infection.

Commensal lactobacilli that produce hydrogen peroxide (H(2)O(2)) inhibit Neisseria gonorrhoeae in vitro, and clinical data suggest that they are associated with a reduced risk of gonorrhea. We precolonized mice with Lactobacillus crispatus and then challenged them with N. gonorrhoeae, to measure the effects of H(2)O(2)-producing lactobacilli on gonococcal infection. We found no difference in the duration of infection or the number of gonococci recovered from untreated mice and mice colonized with L. crispatus. A gonococcal catalase mutant and a catalase, cytochrome C peroxidase mutant exhibited greater susceptibility to L. crispatus in vitro than did wild-type bacteria; however, recovery of these mutants from mice was not affected by L. crispatus. We also found no evidence that utilization of lactobacillus-produced lactate by N. gonorrhoeae balances the detrimental effects of H(2)O(2) during infection. We conclude that the association between lactobacilli and gonococci is complex and may be subject to factors that have not been reproduced in vitro.

Bypass of DNA lesions generated during anticancer treatment with cisplatin by DNA polymerase eta.

DNA polymerase eta (Pol eta) is a eukaryotic lesion bypass polymerase that helps organisms to survive exposure to ultraviolet (UV) radiation, and tumor cells to gain resistance against cisplatin-based chemotherapy. It allows cells to replicate across cross-link lesions such as 1,2-d(GpG) cisplatin adducts (Pt-GG) and UV-induced cis-syn thymine dimers. We present structural and biochemical analysis of how Pol eta copies Pt-GG-containing DNA. The damaged DNA is bound in an open DNA binding rim. Nucleotidyl transfer requires the DNA to rotate into an active conformation, driven by hydrogen bonding of the templating base to the dNTP. For the 3'dG of the Pt-GG, this step is accomplished by a Watson-Crick base pair to dCTP and is biochemically efficient and accurate. In contrast, bypass of the 5'dG of the Pt-GG is less efficient and promiscuous for dCTP and dATP as a result of the presence of the rigid Pt cross-link. Our analysis reveals the set of structural features that enable Pol eta to replicate across strongly distorting DNA lesions.

PNA-based reagents for the direct and site-specific synthesis of thymine dimer lesions in genomic DNA.

An acetophenone containing PNA-based reagent was designed for the direct and site-specific synthesis of a cis-syn thymidine dimer lesion in genomic DNA.