br STAR Methods br Acknowledgments We are grateful to
Acknowledgments We are grateful to Matthias Mayer at ZMBH in Heidelberg for access to his CD-spectrometer and his help in data interpretation. Technical support by Wolfgang Weinig is gratefully acknowledged. This project was financially supported by the European Union as part of the Affinomics consortium (Health-F4-2010-241481) as well as grants of the German Federal Ministry of Education and Research (BMBF; 031A461) and the US National Science Foundation (NSF 1443228) as part of the ERA-Net MirrorBio consortium.
Introduction Interactions between DNA polymerases and DNA ligases have been documented in a number of different eukaryotic systems, both in the context of base-excision repair and single-strand break repair [, , ]. In this MLCK inhibitor peptide 18 study, we report a prokaryotic polymerase-ligase cooperation that functions in the context of DNA end-joining. On their own, DNA ligases can catalyze the end-joining of two DNA fragments with complementary ends and the repair of a single-stranded nick within double stranded DNA. In conjunction with different cooperating proteins, including ones that hold two DNA fragments together or that perform nuclease or polymerase dependent processing, DNA ligases can also end-join non-complementary DNA fragments in processes such as non-homologous end-joining (NHEJ) [4,5]. Both Klenow and Klentaq DNA polymerases (the large fragment domains of the Pol I polymerases from E. coli and T. aquaticus, respectively) show tight binding affinity to DNA fragments with different types of end-structures including 5′ overhangs, 3′ overhangs, and blunt ends . Furthermore, previous studies of the end-joining abilities of Pol I DNA polymerases have shown that both E. coli and T. aquaticus DNA polymerases are able to use discontinuous templates to produce fill-in products between two DNA fragments [, , , ]. This DNA synthesis-dependent end-joining activity has been demonstrated with fragments having fully complementary overlapping ends, with fragments having limited complementary regions that are interior to non-complementary end sequences, and with blunt ended fragments [, , , ]. In vivo evidence for this synthesis-dependent end-joining activity in E. coli has also been described . 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.
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 . Genetic analysis has revealed that there is considerable functional overlap among the DNA ligases encoded by the three LIG genes in nuclear DNA transactions [, , , , , , , , , ]. A mitochondrial version of DNA ligase IIIα (LigIIIα) is generated by alternative translation initiation [, , , ]. In addition, alternative splicing of the LIG3 gene in male germ cells results in LigIIIβ, which has a different C-terminal region than LigIIIα . The steady state level of LigI is frequently elevated in cancer cell lines and tumor samples [, ]. This presumably reflects the hyperproliferative state of cancer cells since LigI is the predominant ligase involved in DNA replication [, , ]. 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 [, , , , ]. 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.