The Roles of UmuD in Regulating Mutagenesis

All organisms are subject to DNA damage from both endogenous and environmental sources. DNA damage that is not fully repaired can lead to mutations. Mutagenesis is now understood to be an active process, in part facilitated by lower-fidelity DNA polymerases that replicate DNA in an error-prone manner. Y-family DNA polymerases, found throughout all domains of life, are characterized by their lower fidelity on undamaged DNA and their specialized ability to copy damaged DNA. TwoE. coliY-family DNA polymerases are responsible for copying damaged DNA as well as for mutagenesis. These DNA polymerases interact with different forms of UmuD, a dynamic protein that regulates mutagenesis. The UmuD gene products, regulated by the SOS response, exist in two principal forms:UmuD2, which prevents mutagenesis, andUmuD2′, which facilitates UV-induced mutagenesis. This paper focuses on the multiple conformations of the UmuD gene products and how their protein interactions regulate mutagenesis.

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Mutagenesis and More: umuDC and the Escherichia coli SOS Response

Genetics ◽

10.1093/genetics/148.4.1599 ◽

1998 ◽

Vol 148 (4) ◽

pp. 1599-1610 ◽

Cited By ~ 10

Author(s):

Bradley T Smith ◽

Graham C Walker

Keyword(s):

Escherichia Coli ◽

Dna Damage ◽

Cellular Response ◽

Sos Response ◽

Posttranslational Regulation ◽

E Coli ◽

Sos Mutagenesis ◽

Induced Mutagenesis ◽

Mechanistic Basis ◽

Increase Cell Survival

Abstract The cellular response to DNA damage that has been most extensively studied is the SOS response of Escherichia coli. Analyses of the SOS response have led to new insights into the transcriptional and posttranslational regulation of processes that increase cell survival after DNA damage as well as insights into DNA-damage-induced mutagenesis, i.e., SOS mutagenesis. SOS mutagenesis requires the recA and umuDC gene products and has as its mechanistic basis the alteration of DNA polymerase III such that it becomes capable of replicating DNA containing miscoding and noncoding lesions. Ongoing investigations of the mechanisms underlying SOS mutagenesis, as well as recent observations suggesting that the umuDC operon may have a role in the regulation of the E. coli cell cycle after DNA damage has occurred, are discussed.

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Multisite SUMOylation restrains DNA polymerase η interactions with DNA damage sites

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013780 ◽

2020 ◽

Vol 295 (25) ◽

pp. 8350-8362 ◽

Cited By ~ 3

Author(s):

Claire Guérillon ◽

Stine Smedegaard ◽

Ivo A. Hendriks ◽

Michael L. Nielsen ◽

Niels Mailand

Keyword(s):

Dna Damage ◽

Dna Polymerase ◽

Feedback Inhibition ◽

Dna Lesions ◽

Nuclear Antigen ◽

Inhibition Mechanism ◽

Proteomic Profiling ◽

Lesion Bypass ◽

Y Family ◽

Damaged Dna

Translesion DNA synthesis (TLS) mediated by low-fidelity DNA polymerases is an essential cellular mechanism for bypassing DNA lesions that obstruct DNA replication progression. However, the access of TLS polymerases to the replication machinery must be kept tightly in check to avoid excessive mutagenesis. Recruitment of DNA polymerase η (Pol η) and other Y-family TLS polymerases to damaged DNA relies on proliferating cell nuclear antigen (PCNA) monoubiquitylation and is regulated at several levels. Using a microscopy-based RNAi screen, here we identified an important role of the SUMO modification pathway in limiting Pol η interactions with DNA damage sites in human cells. We found that Pol η undergoes DNA damage- and protein inhibitor of activated STAT 1 (PIAS1)-dependent polySUMOylation upon its association with monoubiquitylated PCNA, rendering it susceptible to extraction from DNA damage sites by SUMO-targeted ubiquitin ligase (STUbL) activity. Using proteomic profiling, we demonstrate that Pol η is targeted for multisite SUMOylation, and that collectively these SUMO modifications are essential for PIAS1- and STUbL-mediated displacement of Pol η from DNA damage sites. These findings suggest that a SUMO-driven feedback inhibition mechanism is an intrinsic feature of TLS-mediated lesion bypass functioning to curtail the interaction of Pol η with PCNA at damaged DNA to prevent harmful mutagenesis.

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Genetic analysis of DinG-family helicase YoaA and its interaction with replication clamp-loader protein HolC in E. coli

Journal of Bacteriology ◽

10.1128/jb.00228-21 ◽

2021 ◽

Author(s):

Vincent A. Sutera ◽

Thalia H. Sass ◽

Scott E. Leonard ◽

Lingling Wu ◽

David J. Glass ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Tandem Repeats ◽

Genomic Stability ◽

Sos Response ◽

Template Switching ◽

Clamp Loader ◽

E Coli ◽

C Terminus ◽

Domains Of Life

The XP-D/DinG family of DNA helicases contributes to genomic stability in all three domains of life. We investigate here the role of one of these proteins, YoaA, of Escherichia coli . In E. coli , YoaA aids tolerance to the nucleoside azidothymidine (AZT), a DNA replication inhibitor and physically interacts with a subunit of the DNA polymerase III holoenzyme, HolC. We map the residues of YoaA required for HolC interaction to its C-terminus by yeast two-hybrid analysis. We propose that this interaction competes with HolC’s interaction with HolD and the rest of the replisome; YoaA indeed inhibits growth when overexpressed, dependent on this interaction region. By gene fusions we show YoaA is repressed by LexA and induced in response to DNA damage as part of the SOS response. Induction of YoaA by AZT is biphasic with an immediate response after treatment and a slower response that peaks in the late log phase of growth. This growth-phase dependent induction by AZT is not blocked by lexA3 (Ind - ), which normally negates its self-cleavage, implying another means to induce the DNA damage response that responds to the nutritional state of the cell. We propose that YoaA helicase activity increases access to the 3’ nascent strand during replication; consistent with this, YoaA appears to aid removal of potential A-to-T transversion mutations in ndk mutants, which are prone to nucleotide misincorporation. We provide evidence that YoaA and its paralog DinG also may initiate template-switching that leads to deletions between tandem repeats in DNA. IMPORTANCE Maintaining genomic stability is crucial for all living organisms. Replication of DNA frequently encounters barriers that must be removed to complete genome duplication. Balancing DNA synthesis with its repair is critical and not entirely understood at a mechanistic level. The YoaA protein, studied here, is required for certain types of DNA repair and interacts in an alternative manner with proteins that catalyze DNA replication. YoaA is part of the well-studied LexA-regulated response to DNA damage, the SOS response. We describe an unusual feature of its regulation that promotes induction after DNA damage as the culture begins to experience starvation. Replication fork repair integrates both DNA damage and nutritional signals. We also show that YoaA affects genomic stability.

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RETRACTED ARTICLE: The melanocortin signaling cAMP axis accelerates repair and reduces mutagenesis of platinum-induced DNA damage

Scientific Reports ◽

10.1038/s41598-017-12056-5 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 11

Author(s):

Stuart G. Jarrett ◽

Katharine M. Carter ◽

Brent J. Shelton ◽

John A. D’Orazio

Keyword(s):

Dna Damage ◽

Hek293 Cells ◽

Camp Signaling ◽

Melanocyte Stimulating Hormone ◽

Anchoring Protein ◽

Retracted Article ◽

Induced Mutagenesis ◽

Melanocortin Signaling ◽

Camp Induction ◽

Damaged Dna

AbstractUsing primary melanocytes and HEK293 cells, we found that cAMP signaling accelerates repair of bi- and mono-functional platinum-induced DNA damage. Elevating cAMP signaling either by the agonistic MC1R ligand melanocyte stimulating hormone (MSH) or by pharmacologic cAMP induction by forskolin enhanced clearance of intrastrand cisplatin-adducts in melanocytes or MC1R-transfected HEK293 cells. MC1R antagonists human beta-defensin 3 and agouti signaling protein blocked MSH- but not forskolin-mediated enhancement of platinum-induced DNA damage. cAMP-enhanced repair of cisplatin-induced DNA damage was dependent on PKA-mediated phosphorylation of ATR on S435 which promoted ATR’s interaction with the key NER factor xeroderma pigmentosum A (XPA) and facilitated recruitment of an XPA-ATR-pS435 complex to sites of cisplatin DNA damage. Moreover, we developed an oligonucleotide retrieval immunoprecipitation (ORiP) assay using a novel platinated-DNA substrate to establish kinetics of ATR-pS435 and XPA’s associations with cisplatin-damaged DNA. Expression of a non-phosphorylatable ATR-S435A construct or deletion of A kinase-anchoring protein 12 (AKAP12) impeded platinum adduct clearance and prevented cAMP-mediated enhancement of ATR and XPA’s associations with cisplatin-damaged DNA, indicating that ATR phosphorylation at S435 is necessary for cAMP-enhanced repair of platinum-induced damage and protection against cisplatin-induced mutagenesis. These data implicate cAMP signaling as a critical regulator of genomic stability against platinum-induced mutagenesis.

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Ubiquitin-Binding Motifs in REV1 Protein Are Required for Its Role in the Tolerance of DNA Damage

Molecular and Cellular Biology ◽

10.1128/mcb.01118-06 ◽

2006 ◽

Vol 26 (23) ◽

pp. 8892-8900 ◽

Cited By ~ 143

Author(s):

Caixia Guo ◽

Tie-Shan Tang ◽

Marzena Bienko ◽

Joanne L. Parker ◽

Aleksandra B. Bielen ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Tolerance ◽

Binding Motifs ◽

C Terminus ◽

Ubiquitin Binding ◽

Replication Foci ◽

Induced Mutagenesis ◽

Y Family ◽

Precise Role

ABSTRACT REV1 protein is a eukaryotic member of the Y family of DNA polymerases involved in the tolerance of DNA damage by replicative bypass. The precise role(s) of REV1 in this process is not known. Here we show, by using the yeast two-hybrid assay and the glutathione S-transferase pull-down assay, that mouse REV1 can physically interact with ubiquitin. The association of REV1 with ubiquitin requires the ubiquitin-binding motifs (UBMs) located at the C terminus of REV1. The UBMs also mediate the enhanced association between monoubiquitylated PCNA and REV1. In cells exposed to UV radiation, the association of REV1 with replication foci is dependent on functional UBMs. The UBMs of REV1 are shown to contribute to DNA damage tolerance and damage-induced mutagenesis in vivo.

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MsDpo4—a DinB Homolog fromMycobacterium smegmatis—Is an Error-Prone DNA Polymerase That Can Promote G:T and T:G Mismatches

Journal of Nucleic Acids ◽

10.1155/2012/285481 ◽

2012 ◽

Vol 2012 ◽

pp. 1-8 ◽

Cited By ~ 13

Author(s):

Amit Sharma ◽

Deepak T. Nair

Keyword(s):

Mycobacterium Tuberculosis ◽

Dna Polymerase ◽

Environmental Conditions ◽

Mycobacterium Smegmatis ◽

Molecular Level ◽

Dna Polymerases ◽

Adaptive Mutagenesis ◽

E Coli ◽

Nucleotide Incorporation ◽

Y Family

Error-prone DNA synthesis in prokaryotes imparts plasticity to the genome to allow for evolution in unfavorable environmental conditions, and this phenomenon is termed adaptive mutagenesis. At a molecular level, adaptive mutagenesis is mediated by upregulating the expression of specialized error-prone DNA polymerases that generally belong to the Y-family, such as the polypeptide product of thedinBgene in case ofE. coli. However, unlikeE. coli, it has been seen that expression of the homologs ofdinBinMycobacterium tuberculosisare not upregulated under conditions of stress. These studies suggest that DinB homologs inMycobacteriamight not be able to promote mismatches and participate in adaptive mutagenesis. We show that a representative homolog fromMycobacterium smegmatis(MsDpo4) can carry out template-dependent nucleotide incorporation and therefore is a DNA polymerase. In addition, it is seen that MsDpo4 is also capable of misincorporation with a significant ability to promote G:T and T:G mismatches. The frequency of misincorporation for these two mismatches is similar to that exhibited by archaeal and prokaryotic homologs. Overall, our data show that MsDpo4 has the capacity to facilitate transition mutations and can potentially impart plasticity to the genome.

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Identification and Characterization of Thermostable Y-Family DNA Polymerases η, ι, κ and Rev1 From a Lower Eukaryote, Thermomyces lanuginosus

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.778400 ◽

2021 ◽

Vol 8 ◽

Author(s):

Alexandra Vaisman ◽

John P. McDonald ◽

Mallory R. Smith ◽

Sender L. Aspelund ◽

Thomas C. Evans ◽

...

Keyword(s):

Biochemical Characterization ◽

Dna Polymerases ◽

Biochemical Properties ◽

Thermomyces Lanuginosus ◽

Thermophilic Fungi ◽

Domains Of Life ◽

Y Family ◽

Identification And Characterization ◽

Lower Eukaryote

Y-family DNA polymerases (pols) consist of six phylogenetically separate subfamilies; two UmuC (polV) branches, DinB (pol IV, Dpo4, polκ), Rad30A/POLH (polη), and Rad30B/POLI (polι) and Rev1. Of these subfamilies, DinB orthologs are found in all three domains of life; eubacteria, archaea, and eukarya. UmuC orthologs are identified only in bacteria, whilst Rev1 and Rad30A/B orthologs are only detected in eukaryotes. Within eukaryotes, a wide array of evolutionary diversity exists. Humans possess all four Y-family pols (pols η, ι, κ, and Rev1), Schizosaccharomyces pombe has three Y-family pols (pols η, κ, and Rev1), and Saccharomyces cerevisiae only has polη and Rev1. Here, we report the cloning, expression, and biochemical characterization of the four Y-family pols from the lower eukaryotic thermophilic fungi, Thermomyces lanuginosus. Apart from the expected increased thermostability of the T. lanuginosus Y-family pols, their major biochemical properties are very similar to properties of their human counterparts. In particular, both Rad30B homologs (T. lanuginosus and human polɩ) exhibit remarkably low fidelity during DNA synthesis that is template sequence dependent. It was previously hypothesized that higher organisms had acquired this property during eukaryotic evolution, but these observations imply that polι originated earlier than previously known, suggesting a critical cellular function in both lower and higher eukaryotes.

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