Roles of the Y-family DNA polymerase Dbh in accurate replication of the Sulfolobus genome at high temperature

Sakofsky, Cynthia J.; Foster, Patricia L.; Grogan, Dennis W.

doi:10.1016/j.dnarep.2012.01.005

Cited by 20 publications

(30 citation statements)

References 51 publications

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“…One hotspot matches the Dbh deletogenic motif closely (59 GCCCCC on the minus strand), whereas the other lacks the characteristic G (minus-strand 59 ATTTTTT). Both sites generate approximately equal proportions of +1 and 21 frameshifts in vivo (Grogan et al 2001;Sakofsky et al 2012), whereas purified Dbh makes 21 frameshifts almost exclusively (Potapova et al 2002). Thus the deletogenic activity exhibited by Dbh on undamaged DNA in vitro is consistent with some, but not all, of the observed properties of spontaneous mutation at these sites.…”

Section: No Detectable Contribution Of Dbh To Spontaneous Frameshift supporting

confidence: 67%

“…In vitro, Dbh and Dpo4 preferentially insert dC opposite 8-oxo-dG (Gruz et al 2001;Rechkoblit et al 2006;Zang et al 2006;Maxwell and Suo 2012); this predicts that these enzymes would suppress transversion in vivo if recruited to 8-oxo-dG during replication of Sulfolobus chromosomes. Consistent with this prediction, disrupting the dbh gene (Saci_0554) resulted in increased rates of spontaneous G:C-to-T:A transversions in a chromosomal gene (pyrE) where forward mutations can be selected (Sakofsky et al 2012).…”

mentioning

confidence: 54%

“…These two properties combine to argue that the S. acidocaldarius chromosome should be a particularly sensitive detector of Dbh-mediated mutagenesis. At least two hotspots of spontaneous frameshifts occur within the S. acidocaldarius pyrE gene (Grogan et al 2001;Sakofsky et al 2012). One hotspot matches the Dbh deletogenic motif closely (59 GCCCCC on the minus strand), whereas the other lacks the characteristic G (minus-strand 59 ATTTTTT).…”

Section: No Detectable Contribution Of Dbh To Spontaneous Frameshift mentioning

confidence: 99%

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

Sakofsky

Grogan

2015

Genetics

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Hyperthermophilic archaea offer certain advantages as models of genome replication, and Sulfolobus Y-family polymerases Dpo4 (S. solfataricus) and Dbh (S. acidocaldarius) have been studied intensively in vitro as biochemical and structural models of trans-lesion DNA synthesis (TLS). However, the genetic functions of these enzymes have not been determined in the native context of living cells. We developed the first quantitative genetic assays of replication past defined DNA lesions and error-prone motifs in Sulfolobus chromosomes and used them to measure the efficiency and accuracy of bypass in normal and dbh 2 strains of Sulfolobus acidocaldarius. Oligonucleotide-mediated transformation allowed low levels of abasic-site bypass to be observed in S. acidocaldarius and demonstrated that the local sequence context affected bypass specificity; in addition, most erroneous TLS did not require Dbh function. Applying the technique to another common lesion, 7,8-dihydro-8-oxo-deoxyguanosine (8-oxo-dG), revealed an antimutagenic role of Dbh. The efficiency and accuracy of replication past 8-oxo-dG was higher in the presence of Dbh, and up to 90% of the Dbh-dependent events inserted dC. A third set of assays, based on phenotypic reversion, showed no effect of Dbh function on spontaneous 21 frameshifts in mononucleotide tracts in vivo, despite the extremely frequent slippage at these motifs documented in vitro. Taken together, the results indicate that a primary genetic role of Dbh is to avoid mutations at 8-oxo-dG that occur when other Sulfolobus enzymes replicate past this lesion. The genetic evidence that Dbh is recruited to 8-oxo-dG raises questions regarding the mechanism of recruitment, since Sulfolobus spp. have eukaryotic-like replisomes but no ubiquitin.KEYWORDS trans-lesion DNA synthesis (TLS); abasic site; 7,8-dihydro-8-oxo-deoxyguanosine; oligonucleotide-mediated transformation T HE fact that DNA damage occurs in all living cells poses a threat to the accurate replication and partitioning of their genomes. Cells counter this threat with an array of diverse damage-coping systems, some of which repair the DNA, whereas others enable replication to continue past persistent lesions. Lesion bypass, also termed "damage tolerance," can follow various alternatives, which are broadly distinguished as error-free vs. error-prone (Lehmann et al. 2007). The error-free pathways generally use recombinationassociated functions to pair a strand, newly synthesized on intact template, to the damaged DNA strand; the mechanisms for this include transient reversal of the replication fork and strand exchange at gaps left behind the fork (Sale 2012). These mechanisms bypass lesions accurately, although they can promote other forms of genetic instability, such as ectopic recombination between dispersed repeats (Izhar et al. 2013).Error-prone mechanisms of lesion bypass, in contrast, use specialized, trans-lesion synthesis (TLS) polymerases to continue strand elongation using the damaged template; most of these enzymes belong to t...

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Section: No Detectable Contribution Of Dbh To Spontaneous Frameshift supporting

confidence: 67%

mentioning

confidence: 54%

Section: No Detectable Contribution Of Dbh To Spontaneous Frameshift mentioning

confidence: 99%

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

Sakofsky

Grogan

2015

Genetics

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“…This demarcation of the primase/polymerase activities between PrimPol and Pri S probably reflects the additional replication requirements for much larger genomes, such as more regulated laggingstrand synthesis. Although some archaeal species possess canonical Y-family DNA polymerases (Dpo4), specialized in traversing DNA lesions, a recent study has reported that Sulfolobus strains lacking Dpo4 display no increased sensitivity to DNA damaging agents, including UV (32). This strain also exhibited no difference in rates of spontaneous mutagenesis, suggesting that other TLS pathways assist in bypassing replication-blocking lesions.…”

Section: Discussionmentioning

confidence: 95%

Archaeal replicative primases can perform translesion DNA synthesis

Jóźwiakowski

Gholami

Doherty

2015

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…1A; Table 3). This behavior mimics that of spontaneous mutations in both the S. solfataricus and S. acidocaldarius pyrE genes, where similar motifs occur and account for similar slipped-strand events in the corresponding mutational spectra (3,22,23), albeit at much lower rates.…”

Section: Instability Of the Lacs Reporter Gene In Pjlacsmentioning

confidence: 83%

Endogenous Mutagenesis in Recombinant Sulfolobus Plasmids

Sakofsky

Grogan

2013

J Bacteriol

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Low rates of replication errors in chromosomal genes of Sulfolobus spp. demonstrate that these extreme thermoacidophiles can maintain genome integrity in environments with high temperature and low pH. In contrast to this genetic stability, we observed unusually frequent mutation of the ␤-D-glycosidase gene (lacS) of a shuttle plasmid (pJlacS) propagated in Sulfolobus acidocaldarius. The resulting Lac ؊ mutants also grew faster than the Lac ؉ parent, thereby amplifying the impact of the frequent lacS mutations on the population. We developed a mutant accumulation assay and corrections for the effects of copy number and differential growth for this system; the resulting measurements and calculations yielded a corrected rate of 5.1 ؋ 10 ؊4 mutational events at the lacS gene per plasmid replication. Analysis of independent lacS mutants revealed three types of mutations: (i) G·C-to-A·T transitions, (ii) slipped-strand events, and (iii) deletions. These mutations were frequent in plasmid-borne lacS expressed at a high level but not in single-copy lacS in the chromosome or at lower levels of expression in a plasmid. Substitution mutations arose at only two of 12 potential priming sites of the DNA primase of the pRN1 replicon, but nearly all these mutations created nonsense (chain termination) codons. The spontaneous mutation rate of plasmid-borne lacS was 175-fold higher under high-expression than under low-expression conditions. The results suggest that important DNA repair or replication fidelity functions are impaired or overwhelmed in pJlacS, with results analogous to those of the "transcription-associated mutagenesis" seen in bacteria and eukaryotes.T he universal need of organisms to replicate genomes accurately, and in coordination with cell division, has special significance for "extreme" or "hyper-" thermophiles, which grow optimally at temperatures that kill mesophilic organisms and denature their macromolecules (1). The hyperthermophilic archaea which colonize terrestrial and marine hydrothermal systems represent diverse and ancient lineages, and all grow optimally at temperatures that accelerate DNA decomposition reactions by orders of magnitude relative to those in mesophiles (2). The idea that these archaea should have effective strategies of genome protection which compensate for diverse damaging effects of high temperature thus seems logical and is supported by the low rates of replication errors measured in several Sulfolobus isolates (3, 4). The extreme phylogenetic divergence separating archaea from bacteria and eukaryotes (5) further suggests that the strategies which preserve genome integrity in hyperthermophilic archaea may include molecular features not identified in model organisms. This idea remains consistent with the unusual gene inventories and certain biochemical features of DNA metabolism in these archaea (6). The mechanisms that maintain genomic integrity in hyperthermophilic archaea remain challenging to identify in vivo, however. Recent studies, for example, found that several putative ...

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Roles of the Y-family DNA polymerase Dbh in accurate replication of the Sulfolobus genome at high temperature

Cited by 20 publications

References 51 publications

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

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

Archaeal replicative primases can perform translesion DNA synthesis

Endogenous Mutagenesis in Recombinant Sulfolobus Plasmids

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