scholarly journals High-accuracy lagging-strand DNA replication mediated by DNA polymerase dissociation

2018 ◽  
Vol 115 (16) ◽  
pp. 4212-4217 ◽  
Author(s):  
Katarzyna H. Maslowska ◽  
Karolina Makiela-Dzbenska ◽  
Jin-Yao Mo ◽  
Iwona J. Fijalkowska ◽  
Roel M. Schaaper
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Critical Factor ◽  
Chromosomal Dna ◽  
Experimental Systems ◽  
Dna Strands ◽  
Pol Iii ◽  
Error Removal ◽  
Lagging Strand

The fidelity of DNA replication is a critical factor in the rate at which cells incur mutations. Due to the antiparallel orientation of the two chromosomal DNA strands, one strand (leading strand) is replicated in a mostly processive manner, while the other (lagging strand) is synthesized in short sections called Okazaki fragments. A fundamental question that remains to be answered is whether the two strands are copied with the same intrinsic fidelity. In most experimental systems, this question is difficult to answer, as the replication complex contains a different DNA polymerase for each strand, such as, for example, DNA polymerases δ and ε in eukaryotes. Here we have investigated this question in the bacterium Escherichia coli, in which the replicase (DNA polymerase III holoenzyme) contains two copies of the same polymerase (Pol III, the dnaE gene product), and hence the two strands are copied by the same polymerase. Our in vivo mutagenesis data indicate that the two DNA strands are not copied with the same accuracy, and that, remarkably, the lagging strand has the highest fidelity. We postulate that this effect results from the greater dissociative character of the lagging-strand polymerase, which provides additional options for error removal. Our conclusion is strongly supported by results with dnaE antimutator polymerases characterized by increased dissociation rates.

scholarly journals Near-continuously synthesized leading strands inEscherichia coliare broken by ribonucleotide excision

2019 ◽  
Vol 116 (4) ◽  
pp. 1251-1260 ◽  
Author(s):  
Glen E. Cronan ◽  
Elena A. Kouzminova ◽  
Andrei Kuzminov
Keyword(s):  
Dna Replication ◽  
Excision Repair ◽  
Chromosomal Dna ◽  
E Coli ◽  
Okazaki Fragments ◽  
Leading Strand ◽  
Replication Intermediates ◽  
Lagging Strand

In vitro, purified replisomes drive model replication forks to synthesize continuous leading strands, even without ligase, supporting the semidiscontinuous model of DNA replication. However, nascent replication intermediates isolated from ligase-deficientEscherichia colicomprise only short (on average 1.2-kb) Okazaki fragments. It was long suspected that cells replicate their chromosomal DNA by the semidiscontinuous mode observed in vitro but that, in vivo, the nascent leading strand was artifactually fragmented postsynthesis by excision repair. Here, using high-resolution separation of pulse-labeled replication intermediates coupled with strand-specific hybridization, we show that excision-proficientE. coligenerates leading-strand intermediates >10-fold longer than lagging-strand Okazaki fragments. Inactivation of DNA-repair activities, including ribonucleotide excision, further increased nascent leading-strand size to ∼80 kb, while lagging-strand Okazaki fragments remained unaffected. We conclude that in vivo, repriming occurs ∼70× less frequently on the leading versus lagging strands, and that DNA replication inE. coliis effectively semidiscontinuous.

scholarly journals The B subunit of the DNA polymerase alpha-primase complex in Saccharomyces cerevisiae executes an essential function at the initial stage of DNA replication.

1994 ◽  
Vol 14 (2) ◽  
pp. 923-933 ◽  
Author(s):  
M Foiani ◽  
F Marini ◽  
D Gamba ◽  
G Lucchini ◽  
P Plevani
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Cell Viability ◽  
Dna Polymerase ◽  
Chromosomal Dna ◽  
Dna Polymerase Alpha ◽  
B Subunit ◽  
Primase Complex

The four-subunit DNA polymerase alpha-primase complex is unique in its ability to synthesize DNA chains de novo, and some in vitro data suggest its involvement in initiation and elongation of chromosomal DNA replication, although direct in vivo evidence for a role in the initiation reaction is still lacking. The function of the B subunit of the complex is unknown, but the Saccharomyces cerevisiae POL12 gene, which encodes this protein, is essential for cell viability. We have produced different pol12 alleles by in vitro mutagenesis of the cloned gene. The in vivo analysis of our 18 pol12 alleles indicates that the conserved carboxy-terminal two-thirds of the protein contains regions that are essential for cell viability, while the more divergent NH2-terminal portion is partially dispensable. The characterization of the temperature-sensitive pol12-T9 mutant allele demonstrates that the B subunit is required for in vivo DNA synthesis and correct progression through S phase. Moreover, reciprocal shift experiments indicate that the POL12 gene product plays an essential role at the early stage of chromosomal DNA replication, before the hydroxyurea-sensitive step. A model for the role of the B subunit in initiation of DNA replication at an origin is presented.

scholarly journals In Vivo Protein Interactions within theEscherichia coli DNA Polymerase III Core

1998 ◽  
Vol 180 (6) ◽  
pp. 1563-1566 ◽  
Author(s):  
Piotr Jonczyk ◽  
Adrianna Nowicka ◽  
Iwona J. Fijałkowska ◽  
Roel M. Schaaper ◽  
Zygmunt Cieśla
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Physiological Role ◽  
Dna Polymerase Iii ◽  
Mutator Phenotype ◽  
Α Subunit ◽  
Polymerase Iii ◽  
Pol Iii

ABSTRACT The mechanisms that control the fidelity of DNA replication are being investigated by a number of approaches, including detailed kinetic and structural studies. Important tools in these studies are mutant versions of DNA polymerases that affect the fidelity of DNA replication. It has been suggested that proper interactions within the core of DNA polymerase III (Pol III) of Escherichia colicould be essential for maintaining the optimal fidelity of DNA replication (H. Maki and A. Kornberg, Proc. Natl. Acad. Sci. USA 84:4389–4392, 1987). We have been particularly interested in elucidating the physiological role of the interactions between the DnaE (α subunit [possessing DNA polymerase activity]) and DnaQ (ɛ subunit [possessing 3′→5′ exonucleolytic proofreading activity]) proteins. In an attempt to achieve this goal, we have used theSaccharomyces cerevisiae two-hybrid system to analyze specific in vivo protein interactions. In this report, we demonstrate interactions between the DnaE and DnaQ proteins and between the DnaQ and HolE (θ subunit) proteins. We also tested the interactions of the wild-type DnaE and HolE proteins with three well-known mutant forms of DnaQ (MutD5, DnaQ926, and DnaQ49), each of which leads to a strong mutator phenotype. Our results show that the mutD5 anddnaQ926 mutations do not affect the ɛ subunit-α subunit and ɛ subunit-θ subunit interactions. However, thednaQ49 mutation greatly reduces the strength of interaction of the ɛ subunit with both the α and the θ subunits. Thus, the mutator phenotype of dnaQ49 may be the result of an altered conformation of the ɛ protein, which leads to altered interactions within the Pol III core.

scholarly journals Pif1, RPA, and FEN1 modulate the ability of DNA polymerase δ to overcome protein barriers during DNA synthesis

2020 ◽  
Vol 295 (47) ◽  
pp. 15883-15891 ◽  
Author(s):  
Melanie A. Sparks ◽  
Peter M. Burgers ◽  
Roberto Galletto
Keyword(s):  
Transcription Factors ◽  
Dna Replication ◽  
Dna Binding ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Strand Displacement ◽  
Chromosomal Dna ◽  
Dna Polymerase Δ ◽  
Lagging Strand ◽  
Stimulation Of

Successful DNA replication requires carefully regulated mechanisms to overcome numerous obstacles that naturally occur throughout chromosomal DNA. Scattered across the genome are tightly bound proteins, such as transcription factors and nucleosomes, that are necessary for cell function, but that also have the potential to impede timely DNA replication. Using biochemically reconstituted systems, we show that two transcription factors, yeast Reb1 and Tbf1, and a tightly positioned nucleosome, are strong blocks to the strand displacement DNA synthesis activity of DNA polymerase δ. Although the block imparted by Tbf1 can be overcome by the DNA-binding activity of the single-stranded DNA-binding protein RPA, efficient DNA replication through either a Reb1 or a nucleosome block occurs only in the presence of the 5'-3' DNA helicase Pif1. The Pif1-dependent stimulation of DNA synthesis across strong protein barriers may be beneficial during break-induced replication where barriers are expected to pose a problem to efficient DNA bubble migration. However, in the context of lagging strand DNA synthesis, the efficient disruption of a nucleosome barrier by Pif1 could lead to the futile re-replication of newly synthetized DNA. In the presence of FEN1 endonuclease, the major driver of nick translation during lagging strand replication, Pif1-dependent stimulation of DNA synthesis through a nucleosome or Reb1 barrier is prevented. By cleaving the short 5' tails generated during strand displacement, FEN1 eliminates the entry point for Pif1. We propose that this activity would protect the cell from potential DNA re-replication caused by unwarranted Pif1 interference during lagging strand replication.

Prokaryotic DNA replication mechanisms

1987 ◽  
Vol 317 (1187) ◽  
pp. 395-420 ◽  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Kinetic Studies ◽  
Dna Helicase ◽  
Gene 32 ◽  
Dna Template ◽  
Lagging Strand

The three different prokaryotic replication systems that have been most extensively studied use the same basic components for moving a DNA replication fork, even though the individual proteins are different and lack extensive amino acid sequence homology. In the T4 bacteriophage system, the components of the DNA replication complex can be grouped into functional classes as follows: DNA polymerase (gene 43 protein), helix-destabilizing protein (gene 32 protein), polymerase accessory proteins (gene 44/62 and 45 proteins), and primosome proteins (gene 41 DNA helicase and gene 61 RNA primase). DNA synthesis in the in vitro system starts by covalent addition onto the 3'OH end at a random nick on a double-stranded DNA template and proceeds to generate a replication fork that moves at about the in vivo rate, and with approximately the in vivo base-pairing fidelity. DNA is synthesized at the fork in a continuous fashion on the leading strand and in a discontinuous fashion on the lagging strand (generating short Okazaki fragments with 5'-linked pppApCpXpYpZ pentaribonucleotide primers). Kinetic studies reveal that the DNA polymerase molecule on the lagging strand stays associated with the fork as it moves. Therefore the DNA template on the lagging strand must be folded so that the stop site for the synthesis of one Okazaki fragment is adjacent to the start site for the next such fragment, allowing the polymerase and other replication proteins on the lagging strand to recycle.

scholarly journals Fe-S coordination defects in the replicative DNA polymerase delta cause deleterious DNA replication in vivo and subsequent DNA damage in the yeast Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Roland Chanet ◽  
Dorothée Baïlle ◽  
Marie-Pierre Golinelli-Cohen ◽  
Sylvie Riquier ◽  
Olivier Guittet ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Dna Polymerase Delta ◽  
Chromosomal Dna ◽  
Yeast Saccharomyces Cerevisiae ◽  
C Terminus

Abstract B-type eukaryotic polymerases contain a [4Fe-4S] cluster in their C-terminus domain, whose role is not fully understood yet. Among them, DNA polymerase delta (Polδ) plays an essential role in chromosomal DNA replication, mostly during lagging strand synthesis. Previous in vitro work suggested that the Fe-S cluster in Polδ is required for efficient binding of the Pol31 subunit, ensuring stability of the Polδ complex. Here we analyzed the in vivo consequences resulting from an impaired coordination of the Fe-S cluster in Polδ. We show that a single substitution of the very last cysteine coordinating the cluster by a serine is responsible for the generation of massive DNA damage during S phase, leading to checkpoint activation, requirement of homologous recombination for repair, and ultimately to cell death when the repair capacities of the cells are overwhelmed. These data indicate that impaired Fe-S cluster coordination in Polδ is responsible for aberrant replication. More generally, Fe-S in Polδ may be compromised by various stress including anti-cancer drugs. Possible in vivo Polδ Fe-S cluster oxidation and collapse may thus occur, and we speculate this could contribute to induced genomic instability and cell death, comparable to that observed in pol3-13 cells.

scholarly journals The B subunit of the DNA polymerase alpha-primase complex in Saccharomyces cerevisiae executes an essential function at the initial stage of DNA replication

1994 ◽  
Vol 14 (2) ◽  
pp. 923-933
Author(s):  
M Foiani ◽  
F Marini ◽  
D Gamba ◽  
G Lucchini ◽  
P Plevani
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Cell Viability ◽  
Dna Polymerase ◽  
Chromosomal Dna ◽  
Dna Polymerase Alpha ◽  
B Subunit ◽  
Primase Complex

The four-subunit DNA polymerase alpha-primase complex is unique in its ability to synthesize DNA chains de novo, and some in vitro data suggest its involvement in initiation and elongation of chromosomal DNA replication, although direct in vivo evidence for a role in the initiation reaction is still lacking. The function of the B subunit of the complex is unknown, but the Saccharomyces cerevisiae POL12 gene, which encodes this protein, is essential for cell viability. We have produced different pol12 alleles by in vitro mutagenesis of the cloned gene. The in vivo analysis of our 18 pol12 alleles indicates that the conserved carboxy-terminal two-thirds of the protein contains regions that are essential for cell viability, while the more divergent NH2-terminal portion is partially dispensable. The characterization of the temperature-sensitive pol12-T9 mutant allele demonstrates that the B subunit is required for in vivo DNA synthesis and correct progression through S phase. Moreover, reciprocal shift experiments indicate that the POL12 gene product plays an essential role at the early stage of chromosomal DNA replication, before the hydroxyurea-sensitive step. A model for the role of the B subunit in initiation of DNA replication at an origin is presented.

The mutant β E202K sliding clamp protein impairs DNA polymerase III replication activity

2021 ◽  
Author(s):  
Caleb Homiski ◽  
Michelle K. Scotland ◽  
Vignesh M. P. Babu ◽  
Sundari Chodavarapu ◽  
Robert W. Maul ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Genetic Selection ◽  
Cellular Level ◽  
Dna Polymerase Iii ◽  
E Coli ◽  
Polymerase Iii ◽  
Pol Iii

Expression of the E. coli dnaN -encoded β clamp at ≥10-fold higher than chromosomally-expressed levels impedes growth by interfering with DNA replication. We hypothesized that the excess β clamp sequesters the replicative DNA polymerase III (Pol III) to inhibit replication. As a test of this hypothesis, we measured the ability of eight mutant clamps obtained by their inability to impede growth to stimulate Pol III replication in vitro . Compared with the wild type clamp, seven of the mutants were defective, consistent with their elevated cellular levels failing to sequester Pol III. However, the β E202K mutant, which bears a glutamic acid-to-lysine substitution at residue 202 displayed an increased affinity for Pol IIIα and Pol III core (Pol IIIαεθ), suggesting that it could still effectively sequester Pol III. Of interest, β E202K supported in vitro DNA replication by Pol II and Pol IV, but was defective with Pol III. Genetic experiments indicated that the dnaN E202K strain remained proficient in DNA damage-induced mutagenesis, but was modestly induced for SOS and displayed sensitivity to ultraviolet light and methyl methanesulfonate. These results correlate an impaired ability of the mutant β E202K clamp to support Pol III replication in vivo with its in vitro defect in DNA replication. Taken together, our results: (i) support the model that sequestration of Pol III contributes to growth inhibition, (ii) argue for existence of an additional mechanism that contributes to lethality and (iii) suggest that physical and functional interactions of the β clamp with Pol III are more extensive than currently appreciated. IMPORTANCE The β clamp plays critically important roles in managing the actions of multiple proteins at the replication fork. However, we lack a molecular understanding of both how the clamp interacts with these different partners, and the mechanisms by which it manages their respective actions. We previously exploited the finding that an elevated cellular level of the β clamp impedes E. coli growth by interfering with DNA replication. Using a genetic selection method, we obtained novel mutant β clamps that fail to inhibit growth. Their analysis revealed that β E202K is unique among them. Our work offers new insights into how the β clamp interacts with and manages the actions of E. coli DNA polymerases II, III and IV.

scholarly journals Limiting DNA polymerase delta alters replication dynamics and leads to a dependence on checkpoint activation and recombination-mediated DNA repair

2019 ◽  
Author(s):  
Natasha C Koussa ◽  
Duncan J. Smith
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Genome Instability ◽  
Dna Polymerase Delta ◽  
Origin Firing ◽  
A Genome ◽  
Synthesis And Processing ◽  
The Impact ◽  
Lagging Strand

ABSTRACTDNA polymerase delta (Pol δ) plays several essential roles in eukaryotic DNA replication and repair. At the replication fork, Pol δ is responsible for the synthesis and processing of the lagging-strand. At replication origins, Pol δ has been proposed to initiate leading-strand synthesis by extending the first Okazaki fragment. Destabilizing mutations in human Pol δ subunits cause replication stress and syndromic immunodeficiency. Analogously, reduced levels of Pol δ in Saccharomyces cerevisiae lead to pervasive genome instability. Here, we analyze how the depletion of Pol δ impacts replication origin firing and lagging-strand synthesis during replication elongation in vivo in S. cerevisiae. By analyzing nascent lagging-strand products, we observe a genome-wide change in both the establishment and progression of replication. S-phase progression is slowed in Pol δ depletion, with both globally reduced origin firing and slower replication progression. We find that no polymerase other than Pol δ is capable of synthesizing a substantial amount of lagging-strand DNA, even when Pol δ is severely limiting. We also characterize the impact of impaired lagging-strand synthesis on genome integrity and find increased ssDNA and DNA damage when Pol δ is limiting; these defects lead to a strict dependence on checkpoint signaling and resection-mediated repair pathways for cellular viability.SIGNIFICANCE STATEMENTDNA replication in eukaryotes is carried out by the replisome – a multi-subunit complex comprising the enzymatic activities required to generate two intact daughter DNA strands. DNA polymerase delta (Pol δ) is a multi-functional replisome enzyme responsible for synthesis and processing of the lagging-strand. Mutations in Pol δ cause a variety of human diseases: for example, destabilizing mutations lead to immunodeficiency. We titrate the concentration of Pol δ in budding yeast – a simple model eukaryote with conserved DNA replication machinery. We characterize several replication defects associated with Pol δ scarcity. The defects we observe provide insight into how destabilizing Pol δ mutations lead to genome instability.

scholarly journals A mechanism for preventing asymmetric histone segregation onto replicating DNA strands

Science ◽  
2018 ◽  
Vol 361 (6409) ◽  
pp. 1386-1389 ◽  
Author(s):  
Chuanhe Yu ◽  
Haiyun Gan ◽  
Albert Serra-Cardona ◽  
Lin Zhang ◽  
Songlin Gan ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Histone H3 ◽  
Epigenetic Inheritance ◽  
Yeast Cells ◽  
Replication Forks ◽  
Dna Strands ◽  
Leading Strand ◽  
Lagging Strand

How parental histone (H3-H4)2 tetramers, the primary carriers of epigenetic modifications, are transferred onto leading and lagging strands of DNA replication forks for epigenetic inheritance remains elusive. Here we show that parental (H3-H4)2 tetramers are assembled into nucleosomes onto both leading and lagging strands, with a slight preference for lagging strands. The lagging-strand preference increases markedly in budding yeast cells lacking Dpb3 and Dpb4, two subunits of the leading strand DNA polymerase, Pol ε, owing to the impairment of parental (H3-H4)2 transfer to leading strands. Dpb3-Dpb4 binds H3-H4 in vitro and participates in the inheritance of heterochromatin. These results indicate that different proteins facilitate the transfer of parental (H3-H4)2 onto leading versus lagging strands and that Dbp3-Dpb4 plays an important role in this poorly understood process.

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