Assembly and Distributive Action of an Archaeal DNA Polymerase Holoenzyme

Bauer, Robert J.; Wolff, Ian D.; Zuo, Xiaobing; Lin, Hsiang-Kai; Trakselis, Michael A.

doi:10.1016/j.jmb.2013.09.003

Cited by 19 publications

(38 citation statements)

References 78 publications

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“…Following the cloning of the gene for the catalytic subunit, all subsequent work has utilized the recombinant PolB1 catalytic subunit. Therefore, the majority of studies of Sulfolobus PolB1 have been performed with a sub-fraction of a native holoenzyme192021222324252627.…”

Section: Resultsmentioning

confidence: 99%

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Identification and characterization of a heterotrimeric archaeal DNA polymerase holoenzyme

et al. 2017

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Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. This contrasts with the multi-subunit B-family replicative polymerases of eukaryotes. Here we reveal that the highly studied PolB1 from Sulfolobus solfataricus exists as a heterotrimeric complex in cell extracts. Two small subunits, PBP1 and PBP2, associate with distinct surfaces of the larger catalytic subunit and influence the enzymatic properties of the DNA polymerase. Thus, multi-subunit replicative DNA polymerase holoenzymes are present in all three domains of life. We reveal the architecture of the assembly by a combination of cross-linking coupled with mass spectrometry, X-ray crystallography and single-particle electron microscopy. The small subunits stabilize the holoenzyme assembly and the acidic tail of one small subunit mitigates the ability of the enzyme to perform strand-displacement synthesis, with important implications for lagging strand DNA synthesis.

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Section: Resultsmentioning

confidence: 99%

“…Subsequent work in Sulfolobus solfataricus ( Sso ) led to the cloning of gene for the catalytic subunit, revealing it to be a family B DNA polymerase18. The recombinant PolB1 protein possessed activity and has been subsequently investigated by several laboratories in a range of structural and biochemical analyses192021222324252627.…”

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confidence: 99%

Identification and characterization of a heterotrimeric archaeal DNA polymerase holoenzyme

et al. 2017

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“…Conversely, properties observed in vitro raise the possibility that biologically relevant switching could occur via passive exchange among archaeal DNA polymerases, as has been proposed for bacteria. For example, Dpo1, the replicative polymerase of S. solfataricus, disengages from PCNA frequently (Bauer et al 2013), and 8-oxo-dG in the template blocks its progress, but increases the catalytic efficiency of the TLS polymerase Dpo4 (Maxwell and Suo 2012). In this context, the present study demonstrates that genetic analysis of TLS in S. acidocaldarius can provide important functional insight into mutation avoidance, polymerase recruitment, and other aspects of DNA damage tolerance modeled by the enzymes of hyperthermophilic archaea.…”

Section: Discussionmentioning

confidence: 69%

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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“…Blunt DNA and 5'‐overhang DNA showed stimulations in ATPase rates of 8 and 161%, respectively, which demonstrates preferential loading at a 3' primer over nonoverhangs. The preferential suitability of 5'‐overhang DNA is not surprising, because clamps are known to be loaded there during leading and lagging strand replication …”

Section: Resultsmentioning

confidence: 99%

Synthetic polymers as substrates for a DNA‐sliding clamp protein

Dongen¹,

Clerx²,

Boomen³

et al. 2018

Biopolymers

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The clamp protein (gp45) of the DNA polymerase III of the bacteriophage T4 is known to bind to DNA and stay attached to it in order to facilitate the process of DNA copying by the polymerase. As part of a project aimed at developing new biomimetic data‐encoding systems we have investigated the binding of gp45 to synthetic polymers, that is, rigid, helical polyisocyanopeptides. Molecular modelling studies suggest that the clamp protein may interact with the latter polymers. Experiments aimed at verifying these interactions are presented and discussed.

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Assembly and Distributive Action of an Archaeal DNA Polymerase Holoenzyme

Cited by 19 publications

References 78 publications

Identification and characterization of a heterotrimeric archaeal DNA polymerase holoenzyme

Identification and characterization of a heterotrimeric archaeal DNA polymerase holoenzyme

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

Synthetic polymers as substrates for a DNA‐sliding clamp protein

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