Mechanism of DNA Polymerization Catalyzed by <i>Sulfolobus solfataricus</i> P2 DNA Polymerase IV

Fiala, Kevin A.; Suo, Zucai

doi:10.1021/bi035746z

Cited by 112 publications

(261 citation statements)

References 49 publications

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“…PolIV has an extremely low affinity for the naked primer-template substrate and heavily relies on the β clamp (a bacterial functional homolog of PCNA) to load onto DNA [87,88]. In vitro studies indicate that PolIV and its archeal homolog Dpo4 are relatively faithful polymerases at the incorporation step and the low fidelity primarily results from poor discrimination between correct and incorrect incoming nucleotides at the binding stage and the capacity to elongate mismatched primer template, which results in -1 frameshift mutations [89,90]. Hence, PolIV promotes mutagenesis through three distinct mechanisms: replication error, TLS and incorporation of base analogs.…”

Section: Ddt In Saccharomyces Cerevisiaementioning

confidence: 99%

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

Andersen¹,

Xiao³

2007

Cell Res

184

187

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npg In addition to well-defined DNA repair pathways, all living organisms have evolved mechanisms to avoid cell death caused by replication fork collapse at a site where replication is blocked due to disruptive covalent modifications of DNA. The term DNA damage tolerance (DDT) has been employed loosely to include a collection of mechanisms by which cells survive replication-blocking lesions with or without associated genomic instability. Recent genetic analyses indicate that DDT in eukaryotes, from yeast to human, consists of two parallel pathways with one being error-free and another highly mutagenic. Interestingly, in budding yeast, these two pathways are mediated by sequential modifications of the proliferating cell nuclear antigen (PCNA) by two ubiquitination complexes Rad6-Rad18 and Mms2-Ubc13-Rad5. Damage-induced monoubiquitination of PCNA by Rad6-Rad18 promotes translesion synthesis (TLS) with increased mutagenesis, while subsequent polyubiquitination of PCNA at the same K164 residue by Mms2-Ubc13-Rad5 promotes error-free lesion bypass. Data obtained from recent studies suggest that the above mechanisms are conserved in higher eukaryotes. In particular, mammals contain multiple specialized TLS polymerases. Defects in one of the TLS polymerases have been linked to genomic instability and cancer. DNA damage toleranceIn the presence of spontaneous or carcinogen-induced DNA damage, living cells have to maintain and complete DNA synthesis or risk replication fork collapse. Since the process of DNA licensing is to ensure the genome is duplicated once and only once during each cell cycle, stalled or collapsed replication forks may not be able to restart, which often results in double-strand breaks (DSBs) and causes compromised genome integrity or cell death. In addition to highly conserved DNA repair pathways, all living organisms have evolved schemes to ensure continuation of DNA synthesis in the presence of damage. These schemes were originally termed DNA postreplication repair (PRR) due to observations of transient shortened nascent DNA structures following S phase in response to DNA damage. In bacteria and unicellular yeast, these shortened DNA segments can be measured by an alkaline sedimentation assay [1] or directly observed in electron micrographs [2]. In wild-type cells, these truncated DNA segments were restored to full length following a short recovery time. One typical experiment [1] involved the restoration of the nascent strand following UV exposure in nucleotide excision repair (NER)-deficient cells and was originally assumed to represent a mechanism of DNA repair. However, further investigation revealed that, although the nascent fragments were re-annealed, the original UV-induced pyrimidine dimers, which were responsible for the generation of single-strand gaps, often persisted in the genome [3,4]. It was argued that the replication-blocking lesion was not necessarily corrected, but rather transiently bypassed and carried over to the next generation. Perhaps it is more beneficial for the organi...

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Section: Ddt In Saccharomyces Cerevisiaementioning

confidence: 99%

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

Andersen¹,

Xiao³

2007

Cell Res

184

187

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“…All classes of polymerases use the same five-step kinetic scheme for nucleotide incorporation (1)(2)(3)(4)(5)(6). The kinetic mechanism for the RNAdependent RNA polymerase (RdRp 1 ) from poliovirus (3D pol ) is shown in Scheme 1.…”

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confidence: 99%

“…In most cases, the first conformational-change step (step two) is rate-limiting (2,8,9). In one, chemistry (step three) is rate-limiting (10), and in some (e.g., T4 and RB69 DNA polymerases), the rate-limiting step has not been established.…”

mentioning

confidence: 99%

Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases

Castro

Smidansky

Maksimchuk

et al. 2007

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

146

190

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The rate-limiting step for nucleotide incorporation in the presteady state for most nucleic acid polymerases is thought to be a conformational change. As a result, very little information is available on the role of active-site residues in the chemistry of nucleotidyl transfer. For the poliovirus RNA-dependent RNA polymerase (3D pol ), chemistry is partially (Mg 2؉ ) or completely (Mn 2؉ ) rate limiting. Here we show that nucleotidyl transfer depends on two ionizable groups with pK a values of 7.0 or 8.2 and 10.5, depending upon the divalent cation used in the reaction. A solvent deuterium isotope effect of three to seven was observed on the rate constant for nucleotide incorporation in the pre-steady state; none was observed in the steady state. Proton-inventory experiments were consistent with two protons being transferred during the rate-limiting transition state of the reaction, suggesting that both deprotonation of the 3 -hydroxyl nucleophile and protonation of the pyrophosphate leaving group occur in the transition state for phosphodiester bond formation. Importantly, two proton transfers occur in the transition state for nucleotidyl-transfer reactions catalyzed by RB69 DNA-dependent DNA polymerase, T7 DNA-dependent RNA polymerase and HIV reverse transcriptase. Interpretation of these data in the context of known polymerase structures suggests the existence of a general base for deprotonation of the 3 -OH nucleophile, although use of a water molecule cannot be ruled out conclusively, and a general acid for protonation of the pyrophosphate leaving group in all nucleic acid polymerases. These data imply an associative-like transition-state structure.general-acid-base catalysis ͉ phosphoryl transfer ͉ two-metal-ion mechanism N ucleic acid polymerases are essential for the maintenance and expression of the genomes of all organisms. All classes of polymerases use the same five-step kinetic scheme for nucleotide incorporation (1-6). The kinetic mechanism for the RNAdependent RNA polymerase (RdRp 1 ) from poliovirus (3D pol ) is shown in Scheme 1. One of the advantages of this system is that once 3D pol assembles onto the primer-template substrate, this complex has a half-life of Ͼ2 h (7), greatly simplifying kinetic analysis. In step one, the enzyme-nucleic acid complex (ER n ) binds the nucleoside triphosphate forming a ternary complex (ER n NTP).Step two involves a conformational change (*ER n NTP) that orients the triphosphate for catalysis. In step three, nucleotidyl transfer occurs (*ER nϩ1 PP i ), followed by a second conformational-change step (ER nϩ1 PP i ) and pyrophosphate release (ER nϩ1 ).Although the sequence of events occurring during the nucleotide-addition cycle is identical for all polymerases, the ratelimiting step appears to be different. In most cases, the first conformational-change step (step two) is rate-limiting (2, 8, 9). In one, chemistry (step three) is rate-limiting (10), and in some (e.g., T4 and RB69 DNA polymerases), the rate-limiting step has not been established. For 3D pol , bot...

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“…The DNA polymerase Dpo4 from Sulfolobus solfataricus P2 (polymerase IV) (30) has been investigated in considerable detail both in terms of its function (31)(32)(33)(34)(35) and structure (36 -38). Dpo4 can bypass various DNA adducts, including 8-oxoG (39,40), O 6 -MeG (41), O 6 -BzG (42), UV-cross-linked products (43), BPDE adducts (44,45), and abasic lesions (38).…”

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confidence: 99%

Versatility of Y-family Sulfolobus solfataricus DNA Polymerase Dpo4 in Translesion Synthesis Past Bulky N2-Alkylguanine Adducts

Zhang

Eoff

Kozekov

et al. 2009

Journal of Biological Chemistry

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In contrast to replicative DNA polymerases, Sulfolobus solfataricus Dpo4 showed a limited decrease in catalytic efficiency (k cat /K m ) for insertion of dCTP opposite a series of N 2 -alkylguanine templates of increasing size from (methyl (Me) to (9-anthracenyl)-Me (Anth)). Fidelity was maintained with increasing size up to (2-naphthyl)-Me (Naph). The catalytic efficiency increased slightly going from the N 2 -NaphG to the N 2 -AnthG substrate, at the cost of fidelity. Pre-steady-state kinetic bursts were observed for dCTP incorporation throughout the series (N 2 -MeG to N 2 -AnthG), with a decrease in the burst amplitude and k pol , the rate of single-turnover incorporation. The presteady-state kinetic courses with G and all of the six N 2 -alkyl G adducts could be fit to a general DNA polymerase scheme to which was added an inactive complex in equilibrium with the active ternary Dpo4⅐DNA⅐dNTP complex, and only the rates of equilibrium with the inactive complex and phosphodiester bond formation were altered. Two crystal structures of Dpo4 with a template N 2 -NaphG (in a post-insertion register opposite a 3-terminal C in the primer) were solved. One showed N 2 -NaphG in a syn conformation, with the naphthyl group located between the template and the Dpo4 "little finger" domain. The Hoogsteen face was within hydrogen bonding distance of the N4 atoms of the cytosine opposite N 2 -NaphG and the cytosine at the ؊2 position. The second structure showed N 2 -Naph G in an anti conformation with the primer terminus largely disordered. Collectively these results explain the versatility of Dpo4 in bypassing bulky G lesions.

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Mechanism of DNA Polymerization Catalyzed by Sulfolobus solfataricus P2 DNA Polymerase IV

Cited by 112 publications

References 49 publications

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases

Versatility of Y-family Sulfolobus solfataricus DNA Polymerase Dpo4 in Translesion Synthesis Past Bulky N2-Alkylguanine Adducts

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