The purpose of this study was to determine whether oligonucleotides the size of siRNA are permeable to gap junctions and whether a specific siRNA for DNA polymerase β (pol β) can move from one cell to another via gap junctions, thus allowing one cell to inhibit gene expression in another cell directly. To test this hypothesis, fluorescently labelled oligonucleotides (morpholinos) 12, 16 and 24 nucleotides in length were synthesized and introduced into one cell of a pair using a patch pipette. These probes moved from cell to cell through gap junctions composed of connexin 43 (Cx43). Moreover, the rate of transfer declined with increasing length of the oligonucleotide. To test whether siRNA for pol β was permeable to gap junctions we used three cell lines: (1) NRK cells that endogenously express Cx43; (2) Mβ16tsA cells, which express Cx32 and Cx26 but not Cx43; and (3) connexin-deficient N2A cells. NRK and Mβ16tsA cells were each divided into two groups, one of which was stably transfected to express a small hairpin RNA (shRNA), which gives rise to siRNA that targets pol β. These two pol β knockdown cell lines (NRK-kcdc and Mβ16tsA-kcdc) were co-cultured with labelled wild type, NRK-wt or Mβ16tsA-wt cells or N2A cells. The levels of pol β mRNA and protein were determined by semiquantitative RT-PCR and immunoblotting. Co-culture of Mβ16tsA-kcdc cells with Mβ16tsA-wt, N2A or NRK-wt cells had no effect on pol β levels in these cells. Similarly, co-culture of NRK-kcdc with N2A cells had no effect on pol β levels in the N2A cells. In contrast, co-culture of NRK-kcdc with NRK-wt cells resulted in a significant reduction in pol β in the wt cells. The inability of Mβ16tsA-kcdc cells to transfer siRNA is consistent with the fact that oligonucleotides of the 12 nucleotide length were not permeable to Cx32/Cx26 channels. This suggested that Cx43 but not Cx32/Cx26 channels allowed the cell-to-cell movement of the siRNA. These results support the novel hypothesis that non-hybridized and possible hybridized forms of siRNA can move between mammalian cells through connexin-specific gap junctions.

DNA polymerase (pol ) is a member of the X family of DNA polymerases that has been implicated in both base excision repair and non-homologous end joining through in vitro studies. However, to date, no phenotype has been associated with cells deficient in this DNA polymerase. Here we show that pol null mouse fibroblasts are hypersensitive to oxidative DNA damaging agents, suggesting a role of pol in protection of cells against the cytotoxic effects of oxidized DNA. Additionally, pol co-immunoprecipitates with an oxidized base DNA glycosylase, single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1), and localizes to oxidative DNA lesions in situ. From these data, we conclude that pol protects cells against oxidative stress and suggest that it participates in oxidative DNA damage base excision repair.

Base pair mismatches in DNA arise from errors in DNA replication, recombination, and biochemical modification of bases. Mismatches are inherently transient. They are resolved passively by DNA replication, or actively by enzymatic removal and resynthesis of one of the bases. The first step in removal is recognition of strand discontinuity by one of the MutS proteins. Mismatches arising from errors in DNA replication are repaired in favor of the base on the template strand, but other mismatches trigger base excision or nucleotide excision repair (NER), or non-repair pathways such as hypermutation, cell cycle arrest, or apoptosis. We argue that MutL homologues play a key role in determining biologic outcome by recruiting and/or activating effector proteins in response to lesion recognition by MutS. We suggest that the process is regulated by conformational changes in MutL caused by cycles of ATP binding and hydrolysis, and by physiologic changes which influence effector availability.

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