Iron–Sulfur Clusters in DNA Polymerases and Primases of Eukaryotes

Baranovskiy, Andrey G.; Siebler, Hollie M.; Pavlov, Youri I.; Tahirov, T.H.

doi:10.1016/bs.mie.2017.09.003

Cited by 40 publications

(39 citation statements)

References 51 publications

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“…2c, d). Interestingly, the CTDs of Pol α and ε form equivalent complexes with their respective B-subunits but are larger and only coordinate divalent Zn 2+ ions [33][34][35][36][37] . The different size of the CTD and orientation relative to the B-subunit may correlate with the distinct functional dependencies of the three polymerases on PCNA ( Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

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Structure of the processive human Pol δ holoenzyme

et al. 2020

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In eukaryotes, DNA polymerase δ (Pol δ) bound to the proliferating cell nuclear antigen (PCNA) replicates the lagging strand and cooperates with flap endonuclease 1 (FEN1) to process the Okazaki fragments for their ligation. We present the high-resolution cryo-EM structure of the human processive Pol δ-DNA-PCNA complex in the absence and presence of FEN1. Pol δ is anchored to one of the three PCNA monomers through the C-terminal domain of the catalytic subunit. The catalytic core sits on top of PCNA in an open configuration while the regulatory subunits project laterally. This arrangement allows PCNA to thread and stabilize the DNA exiting the catalytic cleft and recruit FEN1 to one unoccupied monomer in a toolbelt fashion. Alternative holoenzyme conformations reveal important functional interactions that maintain PCNA orientation during synthesis. This work sheds light on the structural basis of Pol δ's activity in replicating the human genome.

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Section: Resultsmentioning

confidence: 99%

“…To quantify the presence of Fe-S cluster, the extinction coefficient at 410 nm was monitored 30,32,37,64,65 . It was previously indicated that the protein absorption peak, centered at~280 nm, and the Fe-S absorption peak, centered at~410 nm, are separated enough to be considered independent for quantification analysis 64,65 .…”

Section: Methodsmentioning

confidence: 99%

Structure of the processive human Pol δ holoenzyme

et al. 2020

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“…Eukaryotes have four multimeric PolBs, namely, Alpha (PolAlpha), Delta (PolDelta), Zeta (PolZeta) and Epsilon (PolEpsilon). Each eukaryotic PolB comprises a distinct catalytic PolB subunit (also referred to as A-subunit), a regulatory subunit (B-subunit), and an assortment of accessory subunits ( 2 , 11 ). The four Pols have different functions in the cell.…”

Section: Introductionmentioning

confidence: 99%

“…However, current biochemical and structural data indicates that only CysA motif binds Zn 2+ in all four PolBs. The CysB motif in PolAlpha and PolEpsilon also binds Zn 2+ , but in PolDelta and PolZeta CysB binds the Fe–S cluster ( 11 ). Not surprisingly, CTD structures of PolAlpha and PolEpsilon are considerably more similar to each other than to the corresponding CTDs of PolDelta and PolZeta ( 19–23 ).…”

Section: Introductionmentioning

confidence: 99%

Diversity and evolution of B-family DNA polymerases

Kazlauskas

Krupovìč

Guglielmini

et al. 2020

Nucleic Acids Research

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B-family DNA polymerases (PolBs) represent the most common replicases. PolB enzymes that require RNA (or DNA) primed templates for DNA synthesis are found in all domains of life and many DNA viruses. Despite extensive research on PolBs, their origins and evolution remain enigmatic. Massive accumulation of new genomic and metagenomic data from diverse habitats as well as availability of new structural information prompted us to conduct a comprehensive analysis of the PolB sequences, structures, domain organizations, taxonomic distribution and co-occurrence in genomes. Based on phylogenetic analysis, we identified a new, widespread group of bacterial PolBs that are more closely related to the catalytically active N-terminal half of the eukaryotic PolEpsilon (PolEpsilonN) than to Escherichia coli Pol II. In Archaea, we characterized six new groups of PolBs. Two of them show close relationships with eukaryotic PolBs, the first one with PolEpsilonN, and the second one with PolAlpha, PolDelta and PolZeta. In addition, structure comparisons suggested common origin of the catalytically inactive C-terminal half of PolEpsilon (PolEpsilonC) and PolAlpha. Finally, in certain archaeal PolBs we discovered C-terminal Zn-binding domains closely related to those of PolAlpha and PolEpsilonC. Collectively, the obtained results allowed us to propose a scenario for the evolution of eukaryotic PolBs.

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“…This observation argues that the 4Fe-4S cluster present in the N-terminal part of Pol2 [10,11] is not critically involved in the regulation of pol ε's role in the replisome, though it is needed for the maintenance for the proper architecture of active polymerase. UV-mutability in pol2rc-ΔN strains argues that the 4Fe-4S cluster in the catalytic part of pol ε does not play a major role in processes regulating induced mutagenesis, unlike the Fe-S cluster in pol δ and ζ [42,45,46,68]. The difference in UV induced (Fig.…”

Section: Discussionmentioning

confidence: 95%

Compensation for the absence of the catalytically active half of DNA polymerase ε in yeast by positively selected mutations inCDC28gene

Stepchenkova

Zhuk

Cui

et al. 2020

Preprint

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DNA polymerase ε (pol ε) participates in the leading DNA strand synthesis in eukaryotes. The catalytic subunit of this enzyme, Pol2, is a fusion of two ancestral B-family DNA polymerases. Paradoxically, the catalytically active N-terminal pol is dispensable, and an inactive C-terminal pol is essential for yeast cell viability. Despite extensive studies of strains without the active N-terminal part (mutation pol2-16), it is still unclear how they survive and what the mechanism is of rapid recovery of initially miserably growing cells. Slow progress is attributed to the difficultly of obtaining strains with the defect. We designed a robust method for the construction of mutants with only the C-terminal part of Pol2 using allele pol2rc-ΔN optimized for high protein production. Colonies bearing pol2rc-ΔN appear three times sooner than colonies of pol2-16 but exhibit similar growth defects: sensitivity to hydroxyurea, chromosomal instability, and an elevated level of spontaneous mutagenesis. UV-induced mutagenesis is partially affected, it is lower only at high doses in some reporters. The analysis of the genomes of pol2rc-ΔN isolates revealed the prevalence of nonsynonymous mutations suggesting that the growth recovery was a result of evolution by positive selection for better growth fueled by variants produced by the elevated mutation rate. Mutations in the CDC28 gene, the primary regulator of the cell cycle, were repeatedly found in independent clones. Genetic analysis established that cdc28 alleles single-handedly improve the growth of pol2rc-ΔN strains and suppress HU-sensitivity. The affected amino acids are located on the Cdc28 molecule’s surfaces that mediate contacts with cyclins and kinase subunits. Our work establishes the significance of the CDC28 gene for the resilience of replication and predicts that changes in mammalian homologs, cyclin-dependent kinases may play a role in remastering replication to compensate for the defects in the leading strand synthesis by the dedicated polymerase.Author SummaryThe catalytic subunit of the leading strand DNA polymerase ε, Pol2, consists of two halves made of two different ancestral B-family DNA polymerases. Counterintuitively, the catalytically active N-terminal half is dispensable while the inactive C-terminal part is required for viability. The corresponding strains show a severe growth defect, sensitivity to replication inhibitors, chromosomal instability, and elevated spontaneous mutagenesis. Intriguingly, the slow-growing mutant strains rapidly produced fast-growing clones. We discovered that the adaptation to the loss of the catalytic N-terminal part of Pol2 occurs during evolution by positive selection for a better growth fueled by variants produced by elevated mutation rates. Mutations in the cell cycle-dependent kinase gene, CDC28, can single-handedly improve the growth of strains lacking the N-terminal part of Pol2. Our study predicts that changes in mammalian homologs of cyclin-dependent kinases may play a role in response to the defects of active leading strand polymerase.

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Iron–Sulfur Clusters in DNA Polymerases and Primases of Eukaryotes

Cited by 40 publications

References 51 publications

Structure of the processive human Pol δ holoenzyme

Structure of the processive human Pol δ holoenzyme

Diversity and evolution of B-family DNA polymerases

Compensation for the absence of the catalytically active half of DNA polymerase ε in yeast by positively selected mutations inCDC28gene

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