scholarly journals Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation

2015 ◽  
Vol 112 (48) ◽  
pp. E6624-E6633 ◽  
Author(s):  
María Belén Vallerga ◽  
Sabrina F. Mansilla ◽  
María Belén Federico ◽  
Agustina P. Bertolin ◽  
Vanesa Gottifredi
Keyword(s):  
Dna Polymerase ◽  
Uv Irradiation ◽  
Dna Polymerases ◽  
Strand Break ◽  
S Phase ◽  
Exonuclease Activity ◽  
Replication Forks ◽  
Translesion Dna Synthesis ◽  
Phase Progression ◽  
Uv Lesions

After UV irradiation, DNA polymerases specialized in translesion DNA synthesis (TLS) aid DNA replication. However, it is unclear whether other mechanisms also facilitate the elongation of UV-damaged DNA. We wondered if Rad51 recombinase (Rad51), a factor that escorts replication forks, aids replication across UV lesions. We found that depletion of Rad51 impairs S-phase progression and increases cell death after UV irradiation. Interestingly, Rad51 and the TLS polymerase polη modulate the elongation of nascent DNA in different ways, suggesting that DNA elongation after UV irradiation does not exclusively rely on TLS events. In particular, Rad51 protects the DNA synthesized immediately before UV irradiation from degradation and avoids excessive elongation of nascent DNA after UV irradiation. In Rad51-depleted samples, the degradation of DNA was limited to the first minutes after UV irradiation and required the exonuclease activity of the double strand break repair nuclease (Mre11). The persistent dysregulation of nascent DNA elongation after Rad51 knockdown required Mre11, but not its exonuclease activity, and PrimPol, a DNA polymerase with primase activity. By showing a crucial contribution of Rad51 to the synthesis of nascent DNA, our results reveal an unanticipated complexity in the regulation of DNA elongation across UV-damaged templates.

scholarly journals The Meiosis-Specific Crs1 Cyclin Is Required for Efficient S-Phase Progression and Stable Nuclear Architecture

2021 ◽  
Vol 22 (11) ◽  
pp. 5483
Author(s):  
Luisa F. Bustamante-Jaramillo ◽  
Celia Ramos ◽  
Cristina Martín-Castellanos
Keyword(s):  
Cell Cycle ◽  
Translational Control ◽  
Cell Cycle Progression ◽  
Nuclear Architecture ◽  
Strand Break ◽  
Spindle Pole ◽  
S Phase ◽  
Cycle Progression ◽  
Single Wave ◽  
Phase Progression

Cyclins and CDKs (Cyclin Dependent Kinases) are key players in the biology of eukaryotic cells, representing hubs for the orchestration of physiological conditions with cell cycle progression. Furthermore, as in the case of meiosis, cyclins and CDKs have acquired novel functions unrelated to this primal role in driving the division cycle. Meiosis is a specialized developmental program that ensures proper propagation of the genetic information to the next generation by the production of gametes with accurate chromosome content, and meiosis-specific cyclins are widespread in evolution. We have explored the diversification of CDK functions studying the meiosis-specific Crs1 cyclin in fission yeast. In addition to the reported role in DSB (Double Strand Break) formation, this cyclin is required for meiotic S-phase progression, a canonical role, and to maintain the architecture of the meiotic chromosomes. Crs1 localizes at the SPB (Spindle Pole Body) and is required to stabilize the cluster of telomeres at this location (bouquet configuration), as well as for normal SPB motion. In addition, Crs1 exhibits CDK(Cdc2)-dependent kinase activity in a biphasic manner during meiosis, in contrast to a single wave of protein expression, suggesting a post-translational control of its activity. Thus, Crs1 displays multiple functions, acting both in cell cycle progression and in several key meiosis-specific events.

scholarly journals A Coordinated Temporal Interplay of Nucleosome Reorganization Factor, Sister Chromatin Cohesion Factor, and DNA Polymerase α Facilitates DNA Replication

2004 ◽  
Vol 24 (21) ◽  
pp. 9568-9579 ◽  
Author(s):  
Yanjiao Zhou ◽  
Teresa S.-F. Wang
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
S Phase ◽  
Terminal Region ◽  
Replication Complex ◽  
Dna Polymerase Α ◽  
Chromatid Cohesion ◽  
Phase Progression

ABSTRACT DNA replication depends critically upon chromatin structure. Little is known about how the replication complex overcomes the nucleosome packages in chromatin during DNA replication. To address this question, we investigate factors that interact in vivo with the principal initiation DNA polymerase, DNA polymerase α (Polα). The catalytic subunit of budding yeast Polα (Pol1p) has been shown to associate in vitro with the Spt16p-Pob3p complex, a component of the nucleosome reorganization system required for both replication and transcription, and with a sister chromatid cohesion factor, Ctf4p. Here, we show that an N-terminal region of Polα (Pol1p) that is evolutionarily conserved among different species interacts with Spt16p-Pob3p and Ctf4p in vivo. A mutation in a glycine residue in this N-terminal region of POL1 compromises the ability of Pol1p to associate with Spt16p and alters the temporal ordered association of Ctf4p with Pol1p. The compromised association between the chromatin-reorganizing factor Spt16p and the initiating DNA polymerase Pol1p delays the Pol1p assembling onto and disassembling from the late-replicating origins and causes a slowdown of S-phase progression. Our results thus suggest that a coordinated temporal and spatial interplay between the conserved N-terminal region of the Polα protein and factors that are involved in reorganization of nucleosomes and promoting establishment of sister chromatin cohesion is required to facilitate S-phase progression.

scholarly journals Exo1 phosphorylation inhibits exonuclease activity and prevents fork collapse in rad53 mutants independently of the 14-3-3 proteins

2020 ◽  
Vol 48 (6) ◽  
pp. 3053-3070
Author(s):  
Esther C Morafraile ◽  
Alberto Bugallo ◽  
Raquel Carreira ◽  
María Fernández ◽  
Cristina Martín-Castellanos ◽  
...  
Keyword(s):  
Genome Stability ◽  
Nuclease Activity ◽  
S Phase ◽  
Exonuclease Activity ◽  
Replication Forks ◽  
Post Translational Modifications ◽  
Replicative Stress ◽  
Stalled Replication Forks ◽  
Maintain Genome Stability ◽  
Specific Contribution

Abstract The S phase checkpoint is crucial to maintain genome stability under conditions that threaten DNA replication. One of its critical functions is to prevent Exo1-dependent fork degradation, and Exo1 is phosphorylated in response to different genotoxic agents. Exo1 seemed to be regulated by several post-translational modifications in the presence of replicative stress, but the specific contribution of checkpoint-dependent phosphorylation to Exo1 control and fork stability is not clear. We show here that Exo1 phosphorylation is Dun1-independent and Rad53-dependent in response to DNA damage or dNTP depletion, and in both situations Exo1 is similarly phosphorylated at multiple sites. To investigate the correlation between Exo1 phosphorylation and fork stability, we have generated phospho-mimic exo1 alleles that rescue fork collapse in rad53 mutants as efficiently as exo1-nuclease dead mutants or the absence of Exo1, arguing that Rad53-dependent phosphorylation is the mayor requirement to preserve fork stability. We have also shown that this rescue is Bmh1–2 independent, arguing that the 14-3-3 proteins are dispensable for fork stabilization, at least when Exo1 is downregulated. Importantly, our results indicated that phosphorylation specifically inhibits the 5' to 3'exo-nuclease activity, suggesting that this activity of Exo1 and not the flap-endonuclease, is the enzymatic activity responsible of the collapse of stalled replication forks in checkpoint mutants.

scholarly journals DNA polymerase iota and related Rad30–like enzymes

2001 ◽  
Vol 356 (1405) ◽  
pp. 53-60 ◽  
Author(s):  
John P. McDonald ◽  
Agnès Tissier ◽  
Ekaterina G. Frank ◽  
Shigenori Iwai ◽  
Fumio Hanaoka ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Xeroderma Pigmentosum ◽  
Molecular Mechanisms ◽  
Dna Polymerases ◽  
Cellular Functions ◽  
Translesion Dna Synthesis ◽  
Dna Polymerase Iota ◽  
Biochemical Studies ◽  
Template Dna

Until recently, the molecular mechanisms of translesion DNA synthesis (TLS), a process whereby a damaged base is used as a template for continued replication, was poorly understood. This area of scientific research has, however, been revolutionized by the finding that proteins long implicated in TLS are, in fact, DNA polymerases. Members of this so–called UmuC/DinB/Rev1/Rad30 superfamily of polymerases have been identified in prokaryotes, eukaryotes and archaea. Biochemical studies with the highly purified polymerases reveal that some, but not all, can traverse blocking lesions in template DNA. All of them share a common feature, however, in that they exhibit low fidelity when replicating undamaged DNA. Of particular interest to us is the Rad30 subfamily of polymerases found exclusively in eukaryotes. Humans possess two Rad30 paralogs, Rad30A and Rad30B. The RAD30A gene encodes DNA polymerase η and defects in the protein lead to the xeroderma pigmentosum variant (XP–V) phenotype in humans. Very recently RAD30B has also been shown to encode a novel DNA polymerase, designated as Pol ι. Based upon in vitro studies, it appears that Pol ι has the lowest fidelity of any eukaryotic polymerase studied to date and we speculate as to the possible cellular functions of such a remarkably error–prone DNA polymerase.

scholarly journals Multiple deoxyribonucleic acid polymerases from quiescent wheat embryos. Purification and characterization of three enzymes from the soluble cytoplasm and one from purified mitochondria

1979 ◽  
Vol 181 (1) ◽  
pp. 183-191 ◽  
Author(s):  
M Castroviejo ◽  
D Tharaud ◽  
L Tarrago-Litvak ◽  
S Litvak
Keyword(s):  
Dna Polymerase ◽  
Deoxyribonucleic Acid ◽  
Dna Polymerases ◽  
Exonuclease Activity ◽  
Purification And Characterization ◽  
Physico Chemical ◽  
Proof Reading ◽  
Wheat Embryos ◽  
Template Specificity

Three DNA polymerases (A, B and C) have been purified from the soluble cytoplasm of ungerminated embryos. Mainly on the basis of chromatographic, template-specificity and salt-inhibition evidence, we have characterized the three enzymes. Other physico-chemical and enzymic properties are described. From purified mitochondria we have purified a DNA polymerase that behaves like DNA polymerase B on chromatographic and template-specificity criteria. Only highly purified enzyme B from the soluble cytoplasm showed an exonuclease activity able to degrade 3′- or 5′-labelled polydeoxyribonucleotides, as well as a ‘proof-reading’ capacity.

scholarly journals The intra-S phase checkpoint directly regulates replication elongation to preserve the integrity of stalled replisomes

2021 ◽  
Vol 118 (24) ◽  
pp. e2019183118
Author(s):  
Yang Liu ◽  
Lu Wang ◽  
Xin Xu ◽  
Yue Yuan ◽  
Bo Zhang ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Dna Polymerases ◽  
Replication Stress ◽  
Underlying Mechanism ◽  
S Phase ◽  
Replication Forks ◽  
Helicase Activity ◽  
Replicative Helicase ◽  
Checkpoint Kinases ◽  
S Phase Checkpoint

DNA replication is dramatically slowed down under replication stress. The regulation of replication speed is a conserved response in eukaryotes and, in fission yeast, requires the checkpoint kinases Rad3ATR and Cds1Chk2. However, the underlying mechanism of this checkpoint regulation remains unresolved. Here, we report that the Rad3ATR-Cds1Chk2 checkpoint directly targets the Cdc45-MCM-GINS (CMG) replicative helicase under replication stress. When replication forks stall, the Cds1Chk2 kinase directly phosphorylates Cdc45 on the S275, S322, and S397 residues, which significantly reduces CMG helicase activity. Furthermore, in cds1Chk2-mutated cells, the CMG helicase and DNA polymerases are physically separated, potentially disrupting replisomes and collapsing replication forks. This study demonstrates that the intra-S phase checkpoint directly regulates replication elongation, reduces CMG helicase processivity, prevents CMG helicase delinking from DNA polymerases, and therefore helps preserve the integrity of stalled replisomes and replication forks.

scholarly journals Translesion DNA Synthesis Across Lesions Induced by Oxidative Products of Pyrimidines: An Insight into the Mechanism by Microscale Thermophoresis

2019 ◽  
Vol 20 (20) ◽  
pp. 5012 ◽  
Author(s):  
Ondrej Hrabina ◽  
Viktor Brabec ◽  
Olga Novakova
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Dna Duplexes ◽  
Translesion Dna Synthesis ◽  
Processing Enzymes ◽  
Microscale Thermophoresis ◽  
Oxidative Products ◽  
Dna Processing ◽  
Damaged Dna

Oxidative stress in cells can lead to the accumulation of reactive oxygen species and oxidation of DNA precursors. Oxidized nucleotides such as 2’-deoxyribo-5-hydroxyuridin (HdU) and 2’-deoxyribo-5-hydroxymethyluridin (HMdU) can be inserted into DNA during replication and repair. HdU and HMdU have attracted particular interest because they have different effects on damaged-DNA processing enzymes that control the downstream effects of the lesions. Herein, we studied the chemically simulated translesion DNA synthesis (TLS) across the lesions formed by HdU or HMdU using microscale thermophoresis (MST). The thermodynamic changes associated with replication across HdU or HMdU show that the HdU paired with the mismatched deoxyribonucleoside triphosphates disturbs DNA duplexes considerably less than thymidine (dT) or HMdU. Moreover, we also demonstrate that TLS by DNA polymerases across the lesion derived from HdU was markedly less extensive and potentially more mutagenic than that across the lesion formed by HMdU. Thus, DNA polymerization by DNA polymerase η (polη), the exonuclease-deficient Klenow fragment of DNA polymerase I (KF–), and reverse transcriptase from human immunodeficiency virus type 1 (HIV-1 RT) across these pyrimidine lesions correlated with the different stabilization effects of the HdU and HMdU in DNA duplexes revealed by MST. The equilibrium thermodynamic data obtained by MST can explain the influence of the thermodynamic alterations on the ability of DNA polymerases to bypass lesions induced by oxidative products of pyrimidines. The results also highlighted the usefulness of MST in evaluating the impact of oxidative products of pyrimidines on the processing of these lesions by damaged DNA processing enzymes.

scholarly journals Transcriptional Modulator NusA Interacts with Translesion DNA Polymerases in Escherichia coli

2008 ◽  
Vol 191 (2) ◽  
pp. 665-672 ◽  
Author(s):  
Susan E. Cohen ◽  
Veronica G. Godoy ◽  
Graham C. Walker
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Temperature Sensitivity ◽  
Dna Polymerases ◽  
Rna Polymerases ◽  
Translesion Dna Synthesis ◽  
Catalytic Activities ◽  
Translesion Dna

ABSTRACT NusA, a modulator of RNA polymerase, interacts with the DNA polymerase DinB. An increased level of expression of dinB or umuDC suppresses the temperature sensitivity of the nusA11 strain, requiring the catalytic activities of these proteins. We propose that NusA recruits translesion DNA synthesis (TLS) polymerases to RNA polymerases stalled at gaps, coupling TLS to transcription.

scholarly journals Rev1 promotes replication through UV lesions in conjunction with DNA polymerases η, ι, and κ but not DNA polymerase ζ

2015 ◽  
Vol 29 (24) ◽  
pp. 2588-2602 ◽  
Author(s):  
Jung-Hoon Yoon ◽  
Jeseong Park ◽  
Juan Conde ◽  
Maki Wakamiya ◽  
Louise Prakash ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Crucial Role ◽  
Genome Stability ◽  
Dna Polymerases ◽  
Translesion Synthesis ◽  
Dna Lesions ◽  
Human Cells ◽  
Uv Lesions ◽  
Human And Mouse

Translesion synthesis (TLS) DNA polymerases (Pols) promote replication through DNA lesions; however, little is known about the protein factors that affect their function in human cells. In yeast, Rev1 plays a noncatalytic role as an indispensable component of Polζ, and Polζ together with Rev1 mediates a highly mutagenic mode of TLS. However, how Rev1 functions in TLS and mutagenesis in human cells has remained unclear. Here we determined the role of Rev1 in TLS opposite UV lesions in human and mouse fibroblasts and showed that Rev1 is indispensable for TLS mediated by Polη, Polι, and Polκ but is not required for TLS by Polζ. In contrast to its role in mutagenic TLS in yeast, Rev1 promotes predominantly error-free TLS opposite UV lesions in humans. The identification of Rev1 as an indispensable scaffolding component for Polη, Polι, and Polκ, which function in TLS in highly specialized ways opposite a diverse array of DNA lesions and act in a predominantly error-free manner, implicates a crucial role for Rev1 in the maintenance of genome stability in humans.

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