A Role for Yeast and Human Translesion Synthesis DNA Polymerases in Promoting Replication through 3-Methyl Adenine

ABSTRACT 3-Methyl adenine (3meA), a minor-groove DNA lesion, presents a strong block to synthesis by replicative DNA polymerases (Pols). To elucidate the means by which replication through this DNA lesion is mediated in eukaryotic cells, here we carry out genetic studies in the yeast Saccharomyces cerevisiae treated with the alkylating agent methyl methanesulfonate. From the studies presented here, we infer that replication through the 3meA lesion in yeast cells can be mediated by the action of three Rad6-Rad18-dependent pathways that include translesion synthesis (TLS) by Polη or -ζ and an Mms2-Ubc13-Rad5-dependent pathway which presumably operates via template switching. We also express human Pols ι and κ in yeast cells and show that they too can mediate replication through the 3meA lesion in yeast cells, indicating a high degree of evolutionary conservation of the mechanisms that control TLS in yeast and human cells. We discuss these results in the context of previous observations that have been made for the roles of Pols η, ι, and κ in promoting replication through the minor-groove N2-dG adducts.

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Translesion synthesis DNA polymerases promote error-free replication through the minor-groove DNA adduct 3-deaza-3-methyladenine

Journal of Biological Chemistry ◽

10.1074/jbc.m117.808659 ◽

2017 ◽

Vol 292 (45) ◽

pp. 18682-18688 ◽

Cited By ~ 13

Author(s):

Jung-Hoon Yoon ◽

Jayati Roy Choudhury ◽

Jeseong Park ◽

Satya Prakash ◽

Louise Prakash

Keyword(s):

Dna Polymerases ◽

Translesion Synthesis ◽

Minor Groove ◽

Dna Adduct

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Human DNA Polymerase ι Promotes Replication through a Ring-Closed Minor-Groove Adduct That Adopts a syn Conformation in DNA

Molecular and Cellular Biology ◽

10.1128/mcb.25.19.8748-8754.2005 ◽

2005 ◽

Vol 25 (19) ◽

pp. 8748-8754 ◽

Cited By ~ 30

Author(s):

William T. Wolfle ◽

Robert E. Johnson ◽

Irina G. Minko ◽

R. Stephen Lloyd ◽

Satya Prakash ◽

...

Keyword(s):

Dna Polymerase ◽

Dna Polymerases ◽

Minor Groove ◽

Dna Lesions ◽

Unsaturated Aldehyde ◽

Cyclic Form ◽

Human Dna ◽

A Minor ◽

Dna Polymerase Ι

ABSTRACT Acrolein, an α,β-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from oxidation of polyamines. The reaction of acrolein with the N 2 group of guanine in DNA leads to the formation of a cyclic adduct, γ-hydroxy-1,N 2-propano-2′-deoxyguanosine (γ-HOPdG). Previously, we have shown that proficient replication through the γ-HOPdG adduct can be mediated by the sequential action of human DNA polymerases (Pols) ι and κ, in which Polι incorporates either pyrimidine opposite γ-HOPdG, but Polκ extends only from the cytosine. Since γ-HOPdG can adopt either a ring-closed cyclic form or a ring-opened form in DNA, to better understand the mechanisms that Pols ι and κ employ to promote replication through this lesion, we have examined the ability of these polymerases to replicate through the structural analogs of γ-HOPdG that are permanently either ring closed or ring opened. Our studies with these model adducts show that whereas the ring-opened form of γ-HOPdG is not inhibitory to synthesis by human Pols η, ι, or κ, only Polι is able to incorporate nucleotides opposite the ring-closed form, which is known to adopt a syn conformation in DNA. From these studies, we infer that (i) Pols η, ι, and κ have the ability to proficiently replicate through minor-groove DNA lesions that do not perturb the Watson-Crick hydrogen bonding of the template base with the incoming nucleotide, and (ii) Polι can accommodate a minor-groove-adducted template purine which adopts a syn conformation in DNA and forms a Hoogsteen base pair with the incoming nucleotide.

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PCNA Ubiquitylation: Instructive or Permissive to DNA Damage Tolerance Pathways?

Biomolecules ◽

10.3390/biom11101543 ◽

2021 ◽

Vol 11 (10) ◽

pp. 1543

Author(s):

Jun Che ◽

Xin Hong ◽

Hai Rao

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Damage Tolerance ◽

Translesion Synthesis ◽

Dna Lesions ◽

Template Switching ◽

Dna Damage Tolerance ◽

Future Studies ◽

Dna Lesion ◽

Replicative Polymerases

DNA lesions escaping from repair often block the DNA replicative polymerases required for DNA replication and are handled during the S/G2 phases by the DNA damage tolerance (DDT) mechanisms, which include the error-prone translesion synthesis (TLS) and the error-free template switching (TS) pathways. Where the mono-ubiquitylation of PCNA K164 is critical for TLS, the poly-ubiquitylation of the same residue is obligatory for TS. However, it is not known how cells divide the labor between TLS and TS. Due to the fact that the type of DNA lesion significantly influences the TLS and TS choice, we propose that, instead of altering the ratio between the mono- and poly-Ub forms of PCNA, the competition between TLS and TS would automatically determine the selection between the two pathways. Future studies, especially the single integrated lesion “i-Damage” system, would elucidate detailed mechanisms governing the choices of specific DDT pathways.

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Efficient and Error-Free Replication Past a Minor-Groove DNA Adduct by the Sequential Action of Human DNA Polymerases ι and κ

Molecular and Cellular Biology ◽

10.1128/mcb.24.13.5687-5693.2004 ◽

2004 ◽

Vol 24 (13) ◽

pp. 5687-5693 ◽

Cited By ~ 82

Author(s):

M. Todd Washington ◽

Irina G. Minko ◽

Robert E. Johnson ◽

William T. Wolfle ◽

Thomas M. Harris ◽

...

Keyword(s):

Dna Adducts ◽

Dna Polymerases ◽

Minor Groove ◽

Dna Lesions ◽

Initial Reaction ◽

Dna Minor Groove ◽

Nucleotide Incorporation ◽

A Minor ◽

Y Family

ABSTRACT DNA polymerase ι (Polι) is a member of the Y family of DNA polymerases, which promote replication through DNA lesions. The role of Polι in lesion bypass, however, has remained unclear. Polι is highly unusual in that it incorporates nucleotides opposite different template bases with very different efficiencies and fidelities. Since interactions of DNA polymerases with the DNA minor groove provide for the nearly equivalent efficiencies and fidelities of nucleotide incorporation opposite each of the four template bases, we considered the possibility that Polι differs from other DNA polymerases in not being as sensitive to distortions of the minor groove at the site of the incipient base pair and that this enables it to incorporate nucleotides opposite highly distorting minor-groove DNA adducts. To check the validity of this idea, we examined whether Polι could incorporate nucleotides opposite the γ-HOPdG adduct, which is formed from an initial reaction of acrolein with the N2 of guanine. We show here that Polι incorporates a C opposite this adduct with nearly the same efficiency as it does opposite a nonadducted template G residue. The subsequent extension step, however, is performed by Polκ, which efficiently extends from the C incorporated opposite the adduct. Based upon these observations, we suggest that an important biological role of Polι and Polκ is to act sequentially to carry out the efficient and accurate bypass of highly distorting minor-groove DNA adducts of the purine bases.

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Bypass of the Major Alkylative DNA Lesion by Human DNA Polymerase η

Molecules ◽

10.3390/molecules24213928 ◽

2019 ◽

Vol 24 (21) ◽

pp. 3928 ◽

Cited By ~ 3

Author(s):

Myong-Chul Koag ◽

Hunmin Jung ◽

Yi Kou ◽

Seongmin Lee

Keyword(s):

Dna Polymerase ◽

Base Pair ◽

Alkylating Agents ◽

Dna Polymerases ◽

Translesion Synthesis ◽

Catalytic Site ◽

Structural Basis ◽

Dna Lesion ◽

Human Dna ◽

Wide Range

A wide range of endogenous and exogenous alkylating agents attack DNA to generate various alkylation adducts. N7-methyl-2-deoxyguanosine (Fm7dG) is the most abundant alkylative DNA lesion. If not repaired, Fm7dG can undergo spontaneous depurination, imidazole ring-opening, or bypass by translesion synthesis DNA polymerases. Human DNA polymerase η (polη) efficiently catalyzes across Fm7dG in vitro, but its structural basis is unknown. Herein, we report a crystal structure of polη in complex with templating Fm7dG and an incoming nonhydrolyzable dCTP analog, where a 2′-fluorine-mediated transition destabilization approach was used to prevent the spontaneous depurination of Fm7dG. The structure showed that polη readily accommodated the Fm7dG:dCTP base pair with little conformational change of protein and DNA. In the catalytic site, Fm7dG and dCTP formed three hydrogen bonds with a Watson–Crick geometry, indicating that the major keto tautomer of Fm7dG is involved in base pairing. The polη-Fm7dG:dCTP structure was essentially identical to the corresponding undamaged structure, which explained the efficient bypass of the major methylated lesion. Overall, the first structure of translesion synthesis DNA polymerase bypassing Fm7dG suggests that in the catalytic site of Y-family DNA polymerases, small N7-alkylguanine adducts may be well tolerated and form the canonical Watson–Crick base pair with dCTP through their keto tautomers.

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Stoichiometry and compositional plasticity of the yeast nuclear pore complex revealed by quantitative fluorescence microscopy

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1719398115 ◽

2018 ◽

Vol 115 (17) ◽

pp. E3969-E3977 ◽

Cited By ~ 36

Author(s):

Sasikumar Rajoo ◽

Pascal Vallotton ◽

Evgeny Onischenko ◽

Karsten Weis

Keyword(s):

Nuclear Pore Complex ◽

Yeast Cells ◽

Nuclear Pore ◽

Altered Expression ◽

Copy Numbers ◽

Quantitative Image ◽

Multiple Copies ◽

Almost All ◽

High Degree

The nuclear pore complex (NPC) is an eightfold symmetrical channel providing selective transport of biomolecules across the nuclear envelope. Each NPC consists of ∼30 different nuclear pore proteins (Nups) all present in multiple copies per NPC. Significant progress has recently been made in the characterization of the vertebrate NPC structure. However, because of the estimated size differences between the vertebrate and yeast NPC, it has been unclear whether the NPC architecture is conserved between species. Here, we have developed a quantitative image analysis pipeline, termed nuclear rim intensity measurement (NuRIM), to precisely determine copy numbers for almost all Nups within native NPCs of budding yeast cells. Our analysis demonstrates that the majority of yeast Nups are present at most in 16 copies per NPC. This reveals a dramatic difference to the stoichiometry determined for the human NPC, suggesting that despite a high degree of individual Nup conservation, the yeast and human NPC architecture is significantly different. Furthermore, using NuRIM, we examined the effects of mutations on NPC stoichiometry. We demonstrate for two paralog pairs of key scaffold Nups, Nup170/Nup157 and Nup192/Nup188, that their altered expression leads to significant changes in the NPC stoichiometry inducing either voids in the NPC structure or substitution of one paralog by the other. Thus, our results not only provide accurate stoichiometry information for the intact yeast NPC but also reveal an intriguing compositional plasticity of the NPC architecture, which may explain how differences in NPC composition could arise in the course of evolution.

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CRISPR-Associated Primase-Polymerases are implicated in prokaryotic CRISPR-Cas adaptation

Nature Communications ◽

10.1038/s41467-021-23535-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Katerina Zabrady ◽

Matej Zabrady ◽

Peter Kolesar ◽

Arthur W. H. Li ◽

Aidan J. Doherty

Keyword(s):

Dna Repair ◽

Dna Synthesis ◽

Genome Stability ◽

Dna Polymerases ◽

Acquired Immunity ◽

Strand Displacement ◽

Genetic Studies ◽

Type Iiia ◽

Maintain Genome Stability ◽

Cas Proteins

AbstractCRISPR-Cas pathways provide prokaryotes with acquired “immunity” against foreign genetic elements, including phages and plasmids. Although many of the proteins associated with CRISPR-Cas mechanisms are characterized, some requisite enzymes remain elusive. Genetic studies have implicated host DNA polymerases in some CRISPR-Cas systems but CRISPR-specific replicases have not yet been discovered. We have identified and characterised a family of CRISPR-Associated Primase-Polymerases (CAPPs) in a range of prokaryotes that are operonically associated with Cas1 and Cas2. CAPPs belong to the Primase-Polymerase (Prim-Pol) superfamily of replicases that operate in various DNA repair and replication pathways that maintain genome stability. Here, we characterise the DNA synthesis activities of bacterial CAPP homologues from Type IIIA and IIIB CRISPR-Cas systems and establish that they possess a range of replicase activities including DNA priming, polymerisation and strand-displacement. We demonstrate that CAPPs operonically-associated partners, Cas1 and Cas2, form a complex that possesses spacer integration activity. We show that CAPPs physically associate with the Cas proteins to form bespoke CRISPR-Cas complexes. Finally, we propose how CAPPs activities, in conjunction with their partners, may function to undertake key roles in CRISPR-Cas adaptation.

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