scholarly journals DNA polymerase η is a substrate for calpain: A possible mechanism for pol η retention in UV induced replication foci

2021 ◽  
Author(s):  
Jo-Ann Nettersheim ◽  
Régine Janel-Bintz ◽  
Lauriane Kuhn ◽  
Agnès M Cordonnier
Keyword(s):  
Dna Polymerase ◽  
Calcium Ionophore ◽  
Small Subunit ◽  
Dna Lesions ◽  
Calpain Inhibitor ◽  
Translesion Dna Synthesis ◽  
Calcium Dependent ◽  
Catalytically Active ◽  
Radiation Induced ◽  
Replication Foci

DNA polymerase η (pol η) is specifically required for translesion DNA synthesis across ultraviolet radiation-induced DNA lesions. Recruitment of this error-prone DNA polymerase is tightly regulated during replication to avoid mutagenesis and perturbation of fork progression. Here we report that pol η interacts with the calpain small subunit-1 (CAPNS1), in a yeast two-hybrid screening. This interaction is functional as demonstrated by the ability of endogenous calpain to mediate calcium-dependent cleavage of pol η in cell-free extracts and in living cells treated with a calcium ionophore. The proteolysis of pol η is found to occur at position 465 leading to a catalytically active truncated protein containing the PCNA-interacting motif PIP1. Unexpectedly, cell treatment with the specific calpain inhibitor calpeptin results in a decreased extent of pol η foci after UV irradiation, indicating that calpain positively regulates pol η accumulation in replication foci.

scholarly journals The Receptor for Parathyroid Hormone and Parathyroid Hormone-Related Peptide Is Hydrolyzed and Its Signaling Properties Are Altered by Directly Binding the Calpain Small Subunit

Endocrinology ◽  
2005 ◽  
Vol 146 (5) ◽  
pp. 2336-2344 ◽  
Author(s):  
Masako Shimada ◽  
Matthew J. Mahon ◽  
Peter A. Greer ◽  
Gino V. Segre
Keyword(s):  
Parathyroid Hormone ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Small Subunit ◽  
Hek293 Cells ◽  
Calpain Inhibitor ◽  
Dependent Manner ◽  
Intact Cells ◽  
Calcium Dependent

Abstract We show calcium-dependent, direct binding between the N-terminal portion of the PTH/PTHrP receptor (PTH1R) C-terminal intracellular tail and the calpain small subunit. Binding requires, but may not be limited to, amino acids W474, S475, and W477. The wild-type, full-length rat (r) PTH1R, but not rPTH1R with W474A/W477A substitutions, copurifies with the endogenous calpain small subunit in HEK293 cells. Calpain hydrolyzes ΔNt-rPTH1R, a receptor with a 156-amino acid N-terminal deletion, in a calcium-dependent manner in vitro and in intact cells. Most importantly, PTH stimulation increases the cleavage of ΔNt-rPTH1R and rPTH1R-yellow fluorescent protein in HEK293 cells, and of talin in HEK293 cells expressing rPTH1R-yellow fluorescent protein and in ROS17/2.8 osteoblast-like cells that express rPTH1R endogenously. The absence of calpain in Capn4-null embryonic fibroblasts and the lowered calpain activity in MC3T3-E1 osteoblastic cells due to stable expression of the calpain inhibitor, calpastatin, reduce PTH-stimulated cAMP accumulation. The calpain small subunit is the second protein, in addition to the sodium-hydrogen exchanger regulatory factor, and the first enzyme that binds the PTH1R; PTH1R bound to both of these proteins results in altered PTH signaling.

scholarly journals TENT4A Non-Canonical Poly(A) Polymerase Regulates DNA-Damage Tolerance via Multiple Pathways That Are Mutated in Endometrial Cancer

2021 ◽  
Vol 22 (13) ◽  
pp. 6957
Author(s):  
Umakanta Swain ◽  
Gilgi Friedlander ◽  
Urmila Sehrawat ◽  
Avital Sarusi-Portuguez ◽  
Ron Rotkopf ◽  
...  
Keyword(s):  
Endometrial Cancer ◽  
Dna Polymerase ◽  
Mrna Stability ◽  
Antisense Rna ◽  
Bioinformatics Analysis ◽  
Dna Polymerases ◽  
Dna Lesions ◽  
Dna Damage Tolerance ◽  
Translesion Dna Synthesis ◽  
Cancer Genomes

TENT4A (PAPD7) is a non-canonical poly(A) polymerase, of which little is known. Here, we show that TENT4A regulates multiple biological pathways and focuses on its multilayer regulation of translesion DNA synthesis (TLS), in which error-prone DNA polymerases bypass unrepaired DNA lesions. We show that TENT4A regulates mRNA stability and/or translation of DNA polymerase h and RAD18 E3 ligase, which guides the polymerase to replication stalling sites and monoubiquitinates PCNA, thereby enabling recruitment of error-prone DNA polymerases to damaged DNA sites. Remarkably, in addition to the effect on RAD18 mRNA stability via controlling its poly(A) tail, TENT4A indirectly regulates RAD18 via the tumor suppressor CYLD and via the long non-coding antisense RNA PAXIP1-AS2, which had no known function. Knocking down the expression of TENT4A or CYLD, or overexpression of PAXIP1-AS2 led each to reduced amounts of the RAD18 protein and DNA polymerase h, leading to reduced TLS, highlighting PAXIP1-AS2 as a new TLS regulator. Bioinformatics analysis revealed that TLS error-prone DNA polymerase genes and their TENT4A-related regulators are frequently mutated in endometrial cancer genomes, suggesting that TLS is dysregulated in this cancer.

scholarly journals Archaeal replicative primases can perform translesion DNA synthesis

2015 ◽  
Vol 112 (7) ◽  
pp. E633-E638 ◽  
Author(s):  
Stanislaw K. Jozwiakowski ◽  
Farimah Borazjani Gholami ◽  
Aidan J. Doherty
Keyword(s):  
Dna Synthesis ◽  
Uv Light ◽  
Small Subunit ◽  
Dna Lesions ◽  
Cyclobutane Pyrimidine Dimers ◽  
Translesion Dna Synthesis ◽  
Template Strand ◽  
Pyrimidine Dimers ◽  
Translesion Dna ◽  
Y Family

DNA replicases routinely stall at lesions encountered on the template strand, and translesion DNA synthesis (TLS) is used to rescue progression of stalled replisomes. This process requires specialized polymerases that perform translesion DNA synthesis. Although prokaryotes and eukaryotes possess canonical TLS polymerases (Y-family Pols) capable of traversing blocking DNA lesions, most archaea lack these enzymes. Here, we report that archaeal replicative primases (Pri S, primase small subunit) can also perform TLS. Archaeal Pri S can bypass common oxidative DNA lesions, such as 8-Oxo-2'-deoxyguanosines and UV light-induced DNA damage, faithfully bypassing cyclobutane pyrimidine dimers. Although it is well documented that archaeal replicases specifically arrest at deoxyuracils (dUs) due to recognition and binding to the lesions, a replication restart mechanism has not been identified. Here, we report that Pri S efficiently replicates past dUs, even in the presence of stalled replicase complexes, thus providing a mechanism for maintaining replication bypass of these DNA lesions. Together, these findings establish that some replicative primases, previously considered to be solely involved in priming replication, are also TLS proficient and therefore may play important roles in damage tolerance at replication forks.

scholarly journals TENT4A non-canonical poly(A) polymerase regulates DNA-damage tolerance via multiple pathways which are mutated in endometrial cancer

2020 ◽  
Author(s):  
Umakanta Swain ◽  
Gilgi Friedlander ◽  
Urmila Sehrawat ◽  
Avital Sarusi-Portuguez ◽  
Ron Rotkopf ◽  
...  
Keyword(s):  
Endometrial Cancer ◽  
Dna Polymerase ◽  
Mrna Stability ◽  
Antisense Rna ◽  
Bioinformatics Analysis ◽  
Dna Polymerases ◽  
Dna Lesions ◽  
Dna Damage Tolerance ◽  
Translesion Dna Synthesis ◽  
Cancer Genomes

AbstractTENT4A (PAPD7) is a non-canonical poly(A) polymerase, of which little is known. Here we show that TENT4A regulates multiple biological pathways, and focus on its multilayer regulation of translesion DNA synthesis (TLS), in which unrepaired DNA lesions are bypassed by error-prone DNA polymerases. We show that TENT4A regulates mRNA stability and/or translation of DNA polymerase η and RAD18 E3 ligase, which guides the polymerase to replication stalling sites, and monoubiquitinates PCNA, thereby enabling recruitment of error-prone DNA polymerases to damaged DNA sites. Remarkably, in addition to the effect on RAD18 mRNA stability via controlling its poly(A) tail, TENT4A indirectly regulates RAD18 via the tumor suppressor CYLD, and via the long non-coding antisense RNA PAXIP1-AS2, which had no known function. Knocking down the expression of TENT4A or CYLD, or overexpression of PAXIP1-AS2 led each to reduced amounts of the RAD18 protein and DNA polymerase η, leading to reduced TLS, highlighting PAXIP1-AS2 as a new TLS regulator. Bioinformatics analysis revealed that TLS error-prone DNA polymerase genes and their TENT4A-related regulators are frequently mutated in endometrial cancer genomes, suggesting that TLS is dysregulated in this cancer.

scholarly journals DNA polymerase ζ in DNA replication and repair

2019 ◽  
Vol 47 (16) ◽  
pp. 8348-8361 ◽  
Author(s):  
Sara K Martin ◽  
Richard D Wood
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Translesion Synthesis ◽  
Dna Lesions ◽  
Replication Forks ◽  
Dna Replication And Repair ◽  
Radiation Induced ◽  
Break Induced Replication

Abstract Here, we survey the diverse functions of DNA polymerase ζ (pol ζ) in eukaryotes. In mammalian cells, REV3L (3130 residues) is the largest catalytic subunit of the DNA polymerases. The orthologous subunit in yeast is Rev3p. Pol ζ also includes REV7 subunits (encoded by Rev7 in yeast and MAD2L2 in mammalian cells) and two subunits shared with the replicative DNA polymerase, pol δ. Pol ζ is used in response to circumstances that stall DNA replication forks in both yeast and mammalian cells. The best-examined situation is translesion synthesis at sites of covalent DNA lesions such as UV radiation-induced photoproducts. We also highlight recent evidence that uncovers various roles of pol ζ that extend beyond translesion synthesis. For instance, pol ζ is also employed when the replisome operates sub-optimally or at difficult-to-replicate DNA sequences. Pol ζ also participates in repair by microhomology mediated break-induced replication. A rev3 deletion is tolerated in yeast but Rev3l disruption results in embryonic lethality in mice. Inactivation of mammalian Rev3l results in genomic instability and invokes cell death and senescence programs. Targeting of pol ζ function may be a useful strategy in cancer therapy, although chromosomal instability associated with pol ζ deficiency must be considered.

scholarly journals Genetic and Biochemical Characterization of a NovelumuD Mutation: Insights into a Mechanism for UmuD Self-Cleavage

2001 ◽  
Vol 183 (1) ◽  
pp. 347-357 ◽  
Author(s):  
Mark D. Sutton ◽  
Melanie Kim ◽  
Graham C. Walker
Keyword(s):  
Dna Polymerase ◽  
Biochemical Characterization ◽  
Dna Lesions ◽  
Site Directed Mutagenesis ◽  
Translesion Dna Synthesis ◽  
Solution Structures ◽  
Amino Terminal ◽  
Abasic Sites ◽  
Repressor Proteins ◽  
Cyclobutane Dimers

ABSTRACT Most translesion DNA synthesis (TLS) in Escherichia coli is dependent upon the products of the umuDCgenes, which encode a DNA polymerase, DNA polymerase V, with the unique ability to replicate over a variety of DNA lesions, including cyclobutane dimers and abasic sites. The UmuD protein is activated for its role in TLS by a RecA–single-stranded DNA (ssDNA)-facilitated self-cleavage event that serves to remove its amino-terminal 24 residues to yield UmuD′. We have used site-directed mutagenesis to construct derivatives of UmuD and UmuD′ with glycines in place of leucine-101 and arginine-102. These residues are extremely well conserved among the UmuD-like proteins involved in mutagenesis but are poorly conserved among the structurally related LexA-like transcriptional repressor proteins. Based on both the crystal and solution structures of the UmuD′ homodimer, these residues are part of a solvent-exposed loop. Our genetic and biochemical characterizations of these mutant UmuD and UmuD′ proteins indicate that while leucine-101 and arginine-102 are critical for the RecA-ssDNA-facilitated self-cleavage of UmuD, they serve only a minimal role in enabling TLS. These results, and others, suggest that the interaction of RecA-ssDNA with leucine-101 and arginine-102, together with numerous other contacts between UmuD2 and the RecA-ssDNA nucleoprotein filaments, serves to realign lysine-97 relative to serine-60, thereby activating UmuD2 for self-cleavage.

scholarly journals Immunomodulatory Effects of Radiotherapy

2020 ◽  
Vol 21 (21) ◽  
pp. 8151
Author(s):  
Sharda Kumari ◽  
Shibani Mukherjee ◽  
Debapriya Sinha ◽  
Salim Abdisalaam ◽  
Sunil Krishnan ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Carbon Particle ◽  
Dna Lesions ◽  
X Rays ◽  
Adaptive Immune ◽  
High Let Radiation ◽  
Different Types ◽  
High Let ◽  
Radiation Induced ◽  
Tumor Death

Radiation therapy (RT), an integral component of curative treatment for many malignancies, can be administered via an increasing array of techniques. In this review, we summarize the properties and application of different types of RT, specifically, conventional therapy with x-rays, stereotactic body RT, and proton and carbon particle therapies. We highlight how low-linear energy transfer (LET) radiation induces simple DNA lesions that are efficiently repaired by cells, whereas high-LET radiation causes complex DNA lesions that are difficult to repair and that ultimately enhance cancer cell killing. Additionally, we discuss the immunogenicity of radiation-induced tumor death, elucidate the molecular mechanisms by which radiation mounts innate and adaptive immune responses and explore strategies by which we can increase the efficacy of these mechanisms. Understanding the mechanisms by which RT modulates immune signaling and the key players involved in modulating the RT-mediated immune response will help to improve therapeutic efficacy and to identify novel immunomodulatory drugs that will benefit cancer patients undergoing targeted RT.

scholarly journals The SOS Error-Prone DNA Polymerase V Mutasome and β-Sliding Clamp Acting in Concert on Undamaged DNA and during Translesion Synthesis

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1083
Author(s):  
Adhirath Sikand ◽  
Malgorzata Jaszczur ◽  
Linda B. Bloom ◽  
Roger Woodgate ◽  
Michael M. Cox ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Pathogenic Bacteria ◽  
Fold Increase ◽  
Translesion Dna Synthesis ◽  
Sliding Clamp ◽  
Pol V ◽  
Dna Polymerase V ◽  
Acting In Concert

In the mid 1970s, Miroslav Radman and Evelyn Witkin proposed that Escherichia coli must encode a specialized error-prone DNA polymerase (pol) to account for the 100-fold increase in mutations accompanying induction of the SOS regulon. By the late 1980s, genetic studies showed that SOS mutagenesis required the presence of two “UV mutagenesis” genes, umuC and umuD, along with recA. Guided by the genetics, decades of biochemical studies have defined the predicted error-prone DNA polymerase as an activated complex of these three gene products, assembled as a mutasome, pol V Mut = UmuD’2C-RecA-ATP. Here, we explore the role of the β-sliding processivity clamp on the efficiency of pol V Mut-catalyzed DNA synthesis on undamaged DNA and during translesion DNA synthesis (TLS). Primer elongation efficiencies and TLS were strongly enhanced in the presence of β. The results suggest that β may have two stabilizing roles: its canonical role in tethering the pol at a primer-3’-terminus, and a possible second role in inhibiting pol V Mut’s ATPase to reduce the rate of mutasome-DNA dissociation. The identification of umuC, umuD, and recA homologs in numerous strains of pathogenic bacteria and plasmids will ensure the long and productive continuation of the genetic and biochemical journey initiated by Radman and Witkin.

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