The present study demonstrates that E coli

The present study demonstrates that E. coli and Taq Pol I DNA polymerases will also facilitate DNA end-joining activity in the absence of DNA synthesis by enhancing the complementary end-joining activity of E. coli DNA ligase when the polymerases are present at sub-stoichiometric concentrations relative to DNA fragments. The effects of both Pol I polymerases on ligation are specific to E.coli DNA ligase, with neither polymerase able to enhance T4 DNA ligase. We hypothesize that this effect is due to an ability of the Pol I DNA polymerases to stabilize association of the two DNA fragments combined with a productive interaction with E. coli DNA ligase.
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Acknowledgements This work was funded by the NSF Division of Molecular and Cellular Biosciences.
Introduction DNA ligation is required to generate an intact lagging strand during DNA replication as well as in almost every recombination and DNA repair event. In human cells, this reaction is carried out by the DNA ligases encoded by the three human LIG genes, LIG1, LIG3 and LIG4 [1]. Genetic analysis has revealed that there is considerable functional overlap among the DNA ligases encoded by the three LIG genes in nuclear DNA transactions [[2], [3], [4], [5], [6], [7], [8], [9], [10], [11]]. A mitochondrial version of DNA ligase IIIα (LigIIIα) is generated by alternative translation initiation [[12], [13], [14], [15]]. In addition, alternative splicing of the LIG3 gene in male germ NLG919 mg results in LigIIIβ, which has a different C-terminal region than LigIIIα [16]. The steady state level of LigI is frequently elevated in cancer cell lines and tumor samples [[17], [18]]. This presumably reflects the hyperproliferative state of cancer cells since LigI is the predominant ligase involved in DNA replication [[19], [20], [21]]. Unexpectedly, many cancer cell lines exhibit both increased steady state levels of LigIIIα and reduced steady state levels of DNA ligase IV (LigIV), with these reciprocal changes indicative of alterations in the relative contribution of different DNA double-strand break repair pathways between non-malignant and cancer cells [[18], [22], [23], [24], [25]]. The dysregulation of DNA ligases in cancer cells together with the involvement of these enzymes in the repair of DNA damage caused by agents used in cancer chemotherapy and radiation therapy suggests that DNA ligase inhibitors may have utility as cancer therapeutics. A set of small molecule LigI inhibitors were identified through an in silico structure-based screen, using the atomic resolution structure of LigI complexed with nicked DNA [[18], [26]]. This screen yielded inhibitors that were selective for LigI (L82), inhibited both LigI and LigIII (L67) and inhibited all three human DNA ligases (L189). As expected, subtoxic levels of the DNA ligase inhibitors enhanced the cytotoxicity of DNA damaging agents in cancer cell lines [18]. Surprisingly, non-malignant cell lines were not sensitized to DNA damage by the DNA ligase inhibitors under similar conditions, suggesting that there are alterations in genome maintenance pathways between non-malignant and cancer cells [18]. Further studies revealed that the repair of DNA double-strand breaks is abnormal in cancer cells with elevated levels of LigIIIα and PARP1 and that these cells are hypersensitive to inhibitors that target LigIIIα and PARP1 [[22], [23], [25]].