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  • In conclusion we have recombinantly expressed and purified t

    2020-05-22

    In conclusion, we have recombinantly expressed and purified the product of the YP_002262821.1 gene from A. salmonicida and confirmed that it possesses ATP-dependent DNA ligase activity. Comparison of the full-length and truncated versions of the protein indicate that the N-terminal 25 as 481 encode a polypeptide which does not form a functional domain of the enzyme, and based on bioinformatic analysis is most likely a periplasmic localization sequence in the native bacterium. This protein preparation will serve as a basis for more detailed characterization of the enzymatic and structural features of this protein in the future, which will help us to understand its biological function.
    Acknowledgments We would like to thank Prof. Arne Smalås and Dr. Elin Moe for their constructive comments and the Tromsø University Proteomics Platform (TUPP) for technical assistance in protein identification by mass spectrometry. This work was supported by the Research Council of Norway (Grant No. 192123).
    Introduction A serious challenge to genomic integrity is the occurrence of a DNA DSB (double-strand break) [1]. To avoid the pathological outcomes that result from even a single unrepaired DNA DSB, all cells have developed efficient DSB repair pathways. In most organisms, there are two major pathways: HR (homologous recombination) and C-NHEJ (classic-non-homologous end joining) [2], [3]. HR is preferentially used in lower organisms, however in mammals – and particularly in human cells – the majority of DSBs are repaired via C-NHEJ. C-NHEJ facilitates the direct ligation of the broken ends of a DSB. Since the DNA termini formed at DSBs are, however, often complex and can contain non-ligatable end groups, the repair of such DNA lesions may require the processing of the ends prior to ligation [1], [4]. This requirement often leads to the loss or addition of nucleotides from either side of the DSB, making C-NHEJ “error-prone”. The mechanism of C-NHEJ-mediated DSB repair postulates that Ku (the Ku70/Ku86 heterodimer) binds to the DSB ends, where it recruits downstream C-NHEJ factors that facilitate processing [5]. Finally LIGIV (DNA ligase IV), in association with XRCC4 (X-ray-cross-complementation gene 4) and XLF (Cernunnos/XRCC4-like factor), performs the end ligation reaction [1]. This linear, stepwise model for C-NHEJ may be oversimplified as there is evidence that LIGIV, XRCC4 and XLF may perform roles both upstream and downstream in the repair process [6], [7], [8]. There is an additional EJing pathway present in higher eukaryotes. It has interchangeably been referred to as MMEJ (micro-homology-mediated end joining) [9], B-NHEJ (backup-NHEJ) [10] and A-NHEJ (alternative-NHEJ) [11], (hereafter, A-NHEJ). Unlike the HR and C-NHEJ pathways, which are conserved from bacteria to man, the A-NHEJ pathway has evolved in a somewhat checkered manner and can only be detected in about a third of eukaryotic genomes [12]. It is presumed that an end-binding factor besides Ku is required to bind onto the broken DNA ends, stabilize them, protect them from random nuclease degradation and finally funnel the ends into the A-NHEJ pathway [13]. Then, because microhomology is frequently used to mediate the repair event, some end resection is required [14]. Alignment activities to bring the microhomologies into register are also needed, followed by the action of a flap-like nuclease to trim non-base paired regions and finally a ligation complex to covalently link the ends back together [15]. Because the pathway uses microhomology to mediate the repair event, deletions always accompany the repair event, as does loss of one of the blocks of microhomology [4]. Several laboratories have made dedicated attempts to identify A-NHEJ factors. In particular, a brute-force nuclear extract fractionation protocol identified LIGIII (DNA ligase III; [12]), heretofore known only for its role in BER (base excision repair), as the candidate ligase required for A-NHEJ [16]. Using guilt-by-association as a scientific rationale, PARP1 (poly (ADP-ribose) polymerase 1) and XRCC1 (X-ray cross complementing gene 1), two proteins known to interact with LIGIII during BER, were subsequently identified as also being involved in A-NHEJ [13], [17], [18]. PARP1 is presumed to compete with Ku for binding to broken DNA ends thereby dictating pathway choice [13], [18] whereas XRCC1 appears to act as a chaperone for LIGIII [19]. Additional factors have also been implicated in A-NHEJ. Thus, CtIP (C-terminal interacting protein) and the MRN (Mre11:Rad50:Nbs1) complex – factors known to be involved in the end resection events required for HR – have also been implicated in the end resection steps of A-NHEJ [20], [21], [22], [23], [24].