Archaeal replicative primases can perform translesion DNA synthesis

Abstract

DNA replicases routinely stall at lesions encountered on the template strand, and translesion DNA synthesis (TLS) is used to rescue progression of stalled replisomes. This process requires specialized polymerases that perform translesion DNA synthesis. Although prokaryotes and eukaryotes possess canonical TLS polymerases (Y-family Pols) capable of traversing blocking DNA lesions, most archaea lack these enzymes. Here, we report that archaeal replicative primases (Pri S, primase small subunit) can also perform TLS. Archaeal Pri S can bypass common oxidative DNA lesions, such as 8-Oxo-2'-deoxyguanosines and UV light-induced DNA damage, faithfully bypassing cyclobutane pyrimidine dimers. Although it is well documented that archaeal replicases specifically arrest at deoxyuracils (dUs) due to recognition and binding to the lesions, a replication restart mechanism has not been identified. Here, we report that Pri S efficiently replicates past dUs, even in the presence of stalled replicase complexes, thus providing a mechanism for maintaining replication bypass of these DNA lesions. Together, these findings establish that some replicative primases, previously considered to be solely involved in priming replication, are also TLS proficient and therefore may play important roles in damage tolerance at replication forks.archaea | replication | translesion synthesis | AEP | primase T he DNA replication machinery rapidly and accurately copies genomes but is prone to stalling at lesions and physical barriers (1). A variety of cellular pathways have evolved to restart stalled replication forks. These include translesion DNA synthesis (TLS) that is performed by specialized polymerases that synthesize short tracts of DNA opposite lesions, thus enabling reinitiation of replication (2). Error-free bypass mechanisms, mediated by homologous recombination, use an alternative undamaged template to rescue stalled replication forks (3). Stalled replisomes can also be rescued by repriming downstream of the blockage, leaving a gap opposite the lesion (4, 5).Eukaryotes and prokaryotes encode distinct TLS polymerases required for DNA damage tolerance (e.g., Y-family Pols). Although much of our understanding of TLS mechanisms has come from studies of archaeal Y-family DNA polymerases, the majority of archaeal species lack canonical TLS enzymes ( Fig. 1A) (6), surprising given the otherwise high degree of conservation between eukaryotic and archaeal replisomes. Many archaea do not appear to encode nucleotide excision repair or photolyase pathways that remove UV light-induced damage (6). These anomalies pose the question as to how archaea, lacking canonical TLS or lesion repair pathways, tolerate the presence of lesions that stall replication. This is particularly pertinent to archaea because of the harsh environmental conditions under which many species reside, including extreme temperatures, which promote increased levels of DNA damage.Archaeal replicases (B-and D-family Pols) specifically arrest at deoxyuracil (dU) (7,8). This unique featu...

Insight into the Human DNA Primase Interaction with Template-Primer

“…Depending on the organism studied, the functional counterpart of p58 C may be located on the catalytic or a different subunit, may contain an [FeS]-cluster or zinc or none of them, and may work in cis or trans orientation. The proposed common mode of RNA primer binding only at two distant points (the 5Ј and 3Ј ends) is consistent with the significant translesion synthesis activity of DNA primases because they cannot sense the modified nucleotides in the newly synthesized strand (43)(44)(45)(46). Due to this unique mode of RNA primer binding, DNA primases are able to "write and count" but cannot "read what they wrote."…”

Mechanism of Concerted RNA-DNA Primer Synthesis by the Human Primosome

“…The lack of interaction between the human primase and the emerging RNA strand (Fig. 8C) explains the significant ability of DNA primases to perform translesion synthesis (52)(53)(54)(55). The data presented here have wide-ranging therapeutic implications, because they provide new perspectives for the design of specific primosome inhibitors ultimately aimed at arresting the onset of replication in targeted cancer cells.…”

Lesion-Induced Mutation in the Hyperthermophilic Archaeon Sulfolobus acidocaldarius and Its Avoidance by the Y-Family DNA Polymerase Dbh

“…Biochemical studies of the latter enzymes of S. solfataricus indicate limited affinity for DNA substrate, limited processivity, and relatively low rates of polymerization (Choi et al 2011). More recently, the DNA primase of a distantly related hyperthermophilic archaeon was reported to catalyze TLS in vitro (Jozwiakowski et al 2015). In vivo, we observed several distinct types of mutagenesis by non-Dbh enzymes, including OMTassociated base substitution and single-base-pair deletion, and spontaneous frameshift mutation.…”