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  • Our finding that one enzyme acts both as a primase

    2021-07-31

    Our finding that one enzyme acts both as a primase and a DNAP poses evolutionary and mechanistic questions. In particular, why do cellular organisms require two enzymes to fulfill de novo DNA synthesis? The inability of cellular replicases to perform as primases might be dictated by the challenges associated with the binding of the priming 5′ nucleotide, because the active site pocket of a primase has to accommodate the triphosphate moiety, which is substantially more negatively charged compared to the pre-existing primer typically faced by a DNAP. The maintenance of the unstable, short oligonucleotides on the templates is also a considerable challenge that arguably cannot be tackled by processive replicases (Kuchta and Stengel, 2010). We have shown that piPolBs have a unique KTRG motif, alternative to the conserved KxY motif of PolBs, which interacts with the primer terminus (Blasco et al., 1995). Moreover, an invariant lysine nearby the KTRG motif plays a key role both in TLS and de novo primer synthesis (Figure 6). Given the positive charge of this and nearby residues in the piPolB group, it is likely that the extended KH-X8-KTRG motif may induce a highly stable primer terminus-binding mechanism that may favor the binding of the incoming nucleotide and the subsequent stabilization of the ternary complex, which would result in enhanced polymerization capacity. These results establish a structural liaison between TLS and priming capacities of piPolB that should be further explored in the future by structural and biochemical approaches. As mentioned above, all DNA primases lack proofreading capacity. This seems advantageous for the efficient synthesis of short-lived Okazaki fragments. Conversely, the 3′-5′ exonuclease-proofreading activity, which is necessary for faithful DNA replication, could hinder the primase capacity. Thus, piPolB synthetic and degradative activities must be highly coordinated to allow efficient primer synthesis and faithful DNA replication. Furthermore, the piPolB exonuclease activity is also compatible with TLS of non-bulky IOX4 damages, which, as reported previously for pPolB of bacteriophage Bam35 (Berjón-Otero et al., 2015), does not require template strand IOX4 misalignment but tolerates damage-containing mismatches during processive DNA synthesis. The TLS capacity might be of particular importance for mitochondrial pipolins, which, similar to mitochondrial genomes, are likely to be frequently exposed to reactive oxygen species regularly produced during normal mitochondrial respiration (Valentine et al., 1998). Previous studies have shown that replication of pCRY1-like pipolins from fungal mitochondria (Gobbi et al., 1997, Li and Nargang, 1993) can be initiated from multiple origins rather than from a fixed origin (Baidyaroy et al., 2012). However, this observation remained unexplained. In light of our current results, such a replication pattern is consistent with the possibility that pCRY1-like pipolins are replicated by their cognate piPolBs in a primer-independent manner. Analogously, the circular episomal form of bacterial pipolins could be replicated by piPolBs from multiple origins. Replication across bulkier DNA lesions that could not be bypassed by piPolB might benefit from possible downstream re-priming. Replication re-start in UV-exposed E. coli chromosome was suggested in the late 60s (Rupp and Howard-Flanders, 1968), and an origin-independent leading strand re-initiation has been demonstrated experimentally (Heller and Marians, 2006). Accordingly, we have shown that expression of the wild-type piPolB promotes survival of E. coli cells exposed to replication-blocking DNA-damaging agents (Figure 7). Hence, we hypothesize that piPolB might have evolved to maintain pipolins’ DNA by providing faithful and processive de novo DNA replication as well as tolerance to DNA damage, which may also increase the fitness of the host bacteria. The latter mechanism resembles the recently proposed roles of human PrimPol in DNA damage tolerance and bypass (Guilliam and Doherty, 2017, Martínez-Jiménez et al., 2015, Mourón et al., 2013), which in the case of piPolB would be provided by an MGE. According to this hypothesis, pipolins may act as bacterial symbionts contributing to maintenance of the host genome upon genotoxic stress.