RETRACTED ARTICLE: The melanocortin signaling cAMP axis accelerates repair and reduces mutagenesis of platinum-induced DNA damage

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.

Download Full-text

Retracted: AKAP12 mediates PKA-induced phosphorylation of ATR to enhance nucleotide excision repair

Nucleic Acids Research ◽

10.1093/nar/gkw871 ◽

2016 ◽

Vol 44 (22) ◽

pp. 10711-10726 ◽

Cited By ~ 19

Author(s):

Stuart G. Jarrett ◽

Erin M. Wolf Horrell ◽

John A. D'Orazio

Keyword(s):

Dna Damage ◽

Nucleotide Excision Repair ◽

Nuclear Translocation ◽

Excision Repair ◽

Critical Role ◽

Loss Of Function ◽

Melanocortin 1 Receptor ◽

Nucleotide Excision ◽

Anchoring Protein ◽

Induced Mutagenesis

Abstract Loss-of-function in melanocortin 1 receptor (MC1R), a GS protein-coupled receptor that regulates signal transduction through cAMP and protein kinase A (PKA) in melanocytes, is a major inherited melanoma risk factor. Herein, we report a novel cAMP-mediated response for sensing and responding to UV-induced DNA damage regulated by A-kinase-anchoring protein 12 (AKAP12). AKAP12 is identified as a necessary participant in PKA-mediated phosphorylation of ataxia telangiectasia mutated and Rad3-related (ATR) at S435, a post-translational event required for cAMP-enhanced nucleotide excision repair (NER). Moreover, UV exposure promotes ATR-directed phosphorylation of AKAP12 at S732, which promotes nuclear translocation of AKAP12–ATR-pS435. This complex subsequently recruits XPA to UV DNA damage and enhances 5΄ strand incision. Preventing AKAP12's interaction with PKA or with ATR abrogates ATR-pS435 accumulation, delays recruitment of XPA to UV-damaged DNA, impairs NER and increases UV-induced mutagenesis. Our results define a critical role for AKAP12 as an UV-inducible scaffold for PKA-mediated ATR phosphorylation, and identify a repair complex consisting of AKAP12–ATR-pS435-XPA at photodamage, which is essential for cAMP-enhanced NER.

Download Full-text

The Roles of UmuD in Regulating Mutagenesis

Journal of Nucleic Acids ◽

10.4061/2010/947680 ◽

2010 ◽

Vol 2010 ◽

pp. 1-12 ◽

Cited By ~ 8

Author(s):

Jaylene N. Ollivierre ◽

Jing Fang ◽

Penny J. Beuning

Keyword(s):

Dna Damage ◽

Protein Interactions ◽

Dna Polymerases ◽

Gene Products ◽

E Coli ◽

Domains Of Life ◽

Induced Mutagenesis ◽

Multiple Conformations ◽

Y Family ◽

Damaged Dna

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.

Download Full-text

Distinct Calcium Signaling for Wildtype, Loss-of-Function and Gain-of-Function Human MC4R Variants

Journal of the Endocrine Society ◽

10.1210/jendso/bvab048.087 ◽

2021 ◽

Vol 5 (Supplement_1) ◽

pp. A44-A44

Author(s):

Shree S Kumar ◽

Kathleen Grace Mountjoy

Keyword(s):

Energy Balance ◽

Calcium Signaling ◽

Hek293 Cells ◽

Camp Signaling ◽

Loss Of Function ◽

Gain Of Function ◽

Critical Function ◽

Melanocyte Stimulating Hormone ◽

Melanocortin 4 Receptor

Abstract There is compelling evidence for human melanocortin-4-receptor (hMC4R) playing a critical function regulating energy balance; yet signal transduction pathways contributing to this are unclear. The hMC4R activates multiple signaling pathways, including induced increases in cAMP and mobilization of intracellular calcium ([Ca2+]i). Recent evidence showed cAMP signaling was not a good predictor for hMC4R variant-associated obesity. We hypothesize that hMC4R mobilization of [Ca2+]i plays an important role in regulating energy balance. To test this, we developed a robust high-throughput Fura-2 ratiometric fluorescent assay to quantitatively measure [Ca2+]i in vitro. We compared basal and α-melanocyte stimulating hormone (α-MSH) activation of [Ca2+]i for hMC4R-wildtype (WT) and hMC4R-variants stably expressed in HEK293 cells. The loss-of-function variants studied were two obesity-associated variants (R7H and R18L) known to exhibit cAMP signaling similar to WT, two obesity-associated variants (H76R and L250Q) known to exhibit cAMP-constitutive activity (CA) compared to WT, and one overweight-associated variant (H158R) known to exhibit cAMP-CA compared to WT. The gain-of-function variants (V103I and I251L) studied are known to exhibit cAMP signaling similar to WT. The data for basal [Ca2+]i were pooled from three independent experiments performed with WT and all variants in each assay. Data (mean ± SEM) were analyzed using one-way ANOVA with Dunnett’s multiple comparisons. The data (mean ± SEM) for α-MSH activation of hMC4R were pooled from three independent experiments and analyzed using non-parametric sum of squares F-test for maximum best-fit values and EC50. The α-MSH activated assays were performed with each hMC4R variant alongside WT. WT hMC4R and non-CA loss-of-function variants exhibited similar basal and α-MSH activated [Ca2+]i (WT: EC50 = 1.44 nM; R7H: EC50 = 1.40 nM; R18L: EC50 = 1.12 nM). The CA loss-of-function variants exhibited significantly (p < 0.0001) increased basal [Ca2+]i compared with WT (WT = 97.6 ± 0.9 nM; H76R = 114.2 ± 1.7 nM; L250Q = 112.1 ± 2.6 nM; H158R = 110.7 ± 1.8 nM) and significantly lower EC50’s compared with WT (H76R: EC50 = 0.07 nM; p = 0.0019; L250Q: EC50 = 0.09 nM; p = 0.0066; H158R: EC50 = 0.14 nM; p = 0.0009). The gain-of-function hMC4R variants exhibited significantly (p < 0.0001) decreased basal [Ca2+]i compared with WT (WT = 97.6 ± 0.9 nM; V103I: = 86.4 ± 0.9 nM; I251L = 87.5 ± 1.0 nM) and significantly (p = 0.0001) increased α-MSH stimulated maximum [Ca2+]i compared with WT (WT = 224.5 ± 13.6 nM; V103I = 288.2 ± 31.5 nM; I251L = 295.6 ± 20.0 nM). To summarize, we show three distinct patterns of hMC4R-associated calcium signaling; (1) WT and non-CA loss-of-function, (2) CA loss-of-function and (3) non-CA gain-of-function. Future studies are required to understand how hMC4R mobilization of [Ca2+]i might contribute to the regulation of energy balance.

Download Full-text

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.

Download Full-text

Decreased DNA damage by acid and increased repair of acid-damaged DNA in acid-habituatedEscherichia coli

Journal of Applied Bacteriology ◽

10.1111/j.1365-2672.1991.tb02748.x ◽

1991 ◽

Vol 70 (6) ◽

pp. 507-511 ◽

Cited By ~ 38

Author(s):

N. Raja ◽

M. Goodson ◽

D.G. Smith ◽

R.J. Rowbury

Keyword(s):

Dna Damage ◽

Damaged Dna

Download Full-text

Differential FSH Glycosylation Modulates FSHR Oligomerization and Subsequent cAMP Signaling

Frontiers in Endocrinology ◽

10.3389/fendo.2021.765727 ◽

2021 ◽

Vol 12 ◽

Author(s):

Uchechukwu T. Agwuegbo ◽

Emily Colley ◽

Anthony P. Albert ◽

Viktor Y. Butnev ◽

George R. Bousfield ◽

...

Keyword(s):

Super Resolution ◽

Hek293 Cells ◽

Camp Signaling ◽

Camp Production ◽

Functional Roles ◽

Menopausal Women ◽

Resolution Imaging ◽

Post Menopausal

Follicle-stimulating hormone (FSH) and its target G protein-coupled receptor (FSHR) are essential for reproduction. Recent studies have established that the hypo-glycosylated pituitary FSH glycoform (FSH21/18), is more bioactive in vitro and in vivo than the fully-glycosylated variant (FSH24). FSH21/18 predominates in women of reproductive prime and FSH24 in peri-post-menopausal women, suggesting distinct functional roles of these FSH glycoforms. The aim of this study was to determine if differential FSH glycosylation modulated FSHR oligomerization and resulting impact on cAMP signaling. Using a modified super-resolution imaging technique (PD-PALM) to assess FSHR complexes in HEK293 cells expressing FSHR, we observed time and concentration-dependent modulation of FSHR oligomerization by FSH glycoforms. High eFSH and FSH21/18 concentrations rapidly dissociated FSHR oligomers into monomers, whereas FSH24 displayed slower kinetics. The FSHR β-arrestin biased agonist, truncated eLHβ (Δ121-149) combined with asparagine56-deglycosylated eLHα (dg-eLHt), increased FSHR homomerization. In contrast, low FSH21/18 and FSH24 concentrations promoted FSHR association into oligomers. Dissociation of FSHR oligomers correlated with time points where higher cAMP production was observed. Taken together, these data suggest that FSH glycosylation may modulate the kinetics and amplitude of cAMP production, in part, by forming distinct FSHR complexes, highlighting potential avenues for novel therapeutic targeting of the FSHR to improve IVF outcomes.

Download Full-text

Diverse responses to UV light exposure in Acinetobacter include the capacity for DNA damage-induced mutagenesis in the opportunistic pathogens Acinetobacter baumannii and Acinetobacter ursingii

Microbiology ◽

10.1099/mic.0.054668-0 ◽

2012 ◽

Vol 158 (3) ◽

pp. 601-611 ◽

Cited By ~ 22

Author(s):

Janelle M. Hare ◽

James A. Bradley ◽

Ching-li Lin ◽

Tyler J. Elam

Keyword(s):

Dna Damage ◽

Acinetobacter Baumannii ◽

Uv Light ◽

Light Exposure ◽

Opportunistic Pathogens ◽

Induced Mutagenesis

Download Full-text

PARP-1 Activation Directs FUS to DNA Damage Sites to Form PARG-Reversible Compartments Enriched in Damaged DNA

Cell Reports ◽

10.1016/j.celrep.2019.04.031 ◽

2019 ◽

Vol 27 (6) ◽

pp. 1809-1821.e5 ◽

Cited By ~ 33

Author(s):

Anastasia S. Singatulina ◽

Loic Hamon ◽

Maria V. Sukhanova ◽

Bénédicte Desforges ◽

Vandana Joshi ◽

...

Keyword(s):

Dna Damage ◽

Parp 1 ◽

Damaged Dna

Download Full-text

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.

Download Full-text