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

2006 ◽  
Vol 26 (23) ◽  
pp. 8892-8900 ◽  
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.

scholarly journals Novel conserved motifs in Rev1 C-terminus are required for mutagenic DNA damage tolerance

DNA Repair ◽  
2008 ◽  
Vol 7 (9) ◽  
pp. 1455-1470 ◽  
Author(s):  
Sanjay D'Souza ◽  
Lauren S. Waters ◽  
Graham C. Walker
Keyword(s):  
Dna Damage ◽  
Damage Tolerance ◽  
Dna Damage Tolerance ◽  
Conserved Motifs ◽  
C Terminus

scholarly journals DNA Polymerase and dRP-lyase activities of polymorphic variants of human Pol ι

2021 ◽  
Vol 478 (7) ◽  
pp. 1399-1412
Author(s):  
Evgeniy S. Shilkin ◽  
Anastasia S. Gromova ◽  
Margarita P. Smal ◽  
Alena V. Makarova
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Polymerase Activity ◽  
Altered Expression ◽  
Dna Polymerase Iota ◽  
Nucleotide Incorporation ◽  
Lyase Activity ◽  
Polymorphic Variants ◽  
Y Family

Y-family DNA polymerase iota (Pol ι) is involved in DNA damage response and tolerance. Mutations and altered expression level of POLI gene are linked to a higher incidence of cancer. We biochemically characterized five active site polymorphic variants of human Pol ι: R71G (rs3218778), P118L (rs554252419), I236M (rs3218784), E251K (rs3218783) and P365R (rs200852409). We analyzed fidelity of nucleotide incorporation on undamaged DNA, efficiency and accuracy of DNA damage bypass, as well as 5′-deoxyribophosphate lyase (dRP-lyase) activity. The I236M and P118L variants were indistinguishable from the wild-type Pol ι in activity. The E251K and P365R substitutions altered the spectrum of nucleotide incorporation opposite several undamaged DNA bases. The P365R variant also reduced the dRP-lyase activity and possessed the decreased TLS activity opposite 8-oxo-G. The R71G mutation dramatically affected the catalytic activities of Pol ι. The reduced DNA polymerase activity of the R71G variant correlated with an enhanced fidelity of nucleotide incorporation on undamaged DNA, altered lesion-bypass activity and reduced dRP-lyase activity. Therefore, this amino acid substitution likely alters Pol ι functions in vivo.

scholarly journals ArabidopsisImmunophilin-like TWD1 Functionally Interacts with Vacuolar ABC Transporters

2004 ◽  
Vol 15 (7) ◽  
pp. 3393-3405 ◽  
Author(s):  
Markus Geisler ◽  
Marjolaine Girin ◽  
Sabine Brandt ◽  
Vincent Vincenzetti ◽  
Sonia Plaza ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Abc Transporters ◽  
Loss Of Function ◽  
Protein Protein Interactions ◽  
Binding Motifs ◽  
Calmodulin Binding ◽  
C Terminus ◽  
Domain Mapping ◽  
Tetratricopeptide Repeat Domain

Previously, the immunophilin-like protein TWD1 from Arabidopsis has been demonstrated to interact with the ABC transporters AtPGP1 and its closest homologue, AtPGP19. Physiological and biochemical investigation of pgp1/pgp19 and of twd1 plants suggested a regulatory role of TWD1 on AtPGP1/AtPGP19 transport activities. To further understand the dramatic pleiotropic phenotype that is caused by loss-of-function mutation of the TWD1 gene, we were interested in other TWD1 interacting proteins. AtMRP1, a multidrug resistance-associated (MRP/ABCC)-like ABC transporter, has been isolated in a yeast two-hybrid screen. We demonstrate molecular interaction between TWD1 and ABC transporters AtMRP1 and its closest homologue, AtMRP2. Unlike AtPGP1, AtMRP1 binds to the C-terminal tetratricopeptide repeat domain of TWD1, which is well known to mediate protein-protein interactions. Domain mapping proved that TWD1 binds to a motif of AtMRP1 that resembles calmodulin-binding motifs; and calmodulin binding to the C-terminus of MRP1 was verified. By membrane fractionation and GFP-tagging, we localized AtMRP1 to the central vacuolar membrane and the TWD1-AtMRP1 complex was verified in vivo by coimmunoprecipitation. We were able to demonstrate that TWD1 binds to isolated vacuoles and has a significant impact on the uptake of metolachlor-GS and estradiol-β-glucuronide, well-known substrates of vacuolar transporters AtMRP1 and AtMRP2.

scholarly journals Small-molecules that bind to the ubiquitin-binding motif of REV1 inhibit REV1 interaction with K164-monoubiquitinated PCNA and suppress DNA damage tolerance

2018 ◽  
Vol 26 (9) ◽  
pp. 2345-2353 ◽  
Author(s):  
Murugendra Vanarotti ◽  
Benjamin J. Evison ◽  
Marcelo L. Actis ◽  
Akira Inoue ◽  
Ezelle T. McDonald ◽  
...  
Keyword(s):  
Dna Damage ◽  
Small Molecules ◽  
Damage Tolerance ◽  
Binding Motif ◽  
Dna Damage Tolerance ◽  
Ubiquitin Binding

scholarly journals Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Claudia Lancey ◽  
Muhammad Tehseen ◽  
Souvika Bakshi ◽  
Matthew Percival ◽  
Masateru Takahashi ◽  
...  
Keyword(s):  
Dna Damage ◽  
Zinc Fingers ◽  
Structural Basis ◽  
Replication Forks ◽  
Catalytic Core ◽  
Dna Templates ◽  
Secondary Interaction ◽  
Ubiquitin Binding ◽  
Y Family ◽  
Stalled Replication Forks

AbstractY-family DNA polymerase κ (Pol κ) can replicate damaged DNA templates to rescue stalled replication forks. Access of Pol κ to DNA damage sites is facilitated by its interaction with the processivity clamp PCNA and is regulated by PCNA mono-ubiquitylation. Here, we present cryo-EM reconstructions of human Pol κ bound to DNA, an incoming nucleotide, and wild type or mono-ubiquitylated PCNA (Ub-PCNA). In both reconstructions, the internal PIP-box adjacent to the Pol κ Polymerase-Associated Domain (PAD) docks the catalytic core to one PCNA protomer in an angled orientation, bending the DNA exiting the Pol κ active site through PCNA, while Pol κ C-terminal domain containing two Ubiquitin Binding Zinc Fingers (UBZs) is invisible, in agreement with disorder predictions. The ubiquitin moieties are partly flexible and extend radially away from PCNA, with the ubiquitin at the Pol κ-bound protomer appearing more rigid. Activity assays suggest that, when the internal PIP-box interaction is lost, Pol κ is retained on DNA by a secondary interaction between the UBZs and the ubiquitins flexibly conjugated to PCNA. Our data provide a structural basis for the recruitment of a Y-family TLS polymerase to sites of DNA damage.

scholarly journals Division of labor of Y-family polymerases in translesion-DNA synthesis for distinct types of DNA damage

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252587
Author(s):  
Yuriko Inomata ◽  
Takuya Abe ◽  
Masataka Tsuda ◽  
Shunichi Takeda ◽  
Kouji Hirota
Keyword(s):  
Dna Damage ◽  
Dna Synthesis ◽  
Division Of Labor ◽  
Double Knockout ◽  
Translesion Dna Synthesis ◽  
Translesion Dna ◽  
Template Dna ◽  
Y Family ◽  
Higher Sensitivity

Living organisms are continuously under threat from a vast array of DNA-damaging agents, which impact genome DNA. DNA replication machinery stalls at damaged template DNA. The stalled replication fork is restarted via bypass replication by translesion DNA-synthesis polymerases, including the Y-family polymerases Polη, Polι, and Polκ, which possess the ability to incorporate nucleotides opposite the damaged template. To investigate the division of labor among these polymerases in vivo, we generated POLη−/−, POLι−/−, POLκ−/−, double knockout (KO), and triple knockout (TKO) mutants in all combinations from human TK6 cells. TKO cells exhibited a hypersensitivity to ultraviolet (UV), cisplatin (CDDP), and methyl methanesulfonate (MMS), confirming the pivotal role played by these polymerases in bypass replication of damaged template DNA. POLη−/− cells, but not POLι−/− or POLκ−/− cells, showed a strong sensitivity to UV and CDDP, while TKO cells showed a slightly higher sensitivity to UV and CDDP than did POLη−/− cells. On the other hand, TKO cells, but not all single KO cells, exhibited a significantly higher sensitivity to MMS than did wild-type cells. Consistently, DNA-fiber assay revealed that Polη plays a crucial role in bypassing lesions caused by UV-mimetic agent 4-nitroquinoline-1-oxide and CDDP, while all three polymerases play complementary roles in bypassing MMS-induced damage. Our findings indicate that the three Y-family polymerases play distinctly different roles in bypass replication, according to the type of DNA damage generated on the template strand.

scholarly journals A conserved histone H3-H4 interface regulates DNA damage tolerance and homologous recombination during the recovery from replication stress

2021 ◽  
Author(s):  
Masafumi Hayashi ◽  
Kenji Keyamura ◽  
Asami Yoshida ◽  
Mariko Ariyoshi ◽  
Genki Akanuma ◽  
...  
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Damage Tolerance ◽  
Histone H3 ◽  
Wild Type ◽  
Dna Damage Tolerance ◽  
Damage Resistance ◽  
Replication Arrest ◽  
Induced Mutagenesis ◽  
Phase Progression

In eukaryotes, genomic DNA is packaged into nucleosomes, which are the basal components coordinating both the structures and functions of chromatin. Here we screened a collection of mutation for histone H3/H4 mutants in Saccharomyces cerevisiae that affect the DNA damage sensitivity of DNA damage tolerance (DDT)-deficient cells. We identified a class of histone H3/H4 mutations that suppress MMS sensitivity of DDT-deficient cells (hereafter we refer to as the histone SDD mutations), which likely cluster on a specific H3-H4 interface of the nucleosomes. The histone SDD mutations did not suppress the MMS sensitivity of DDT-deficient cells in the absence of Rad51, indicating that homologous recombination (HR) is responsible for DNA damage resistance. Furthermore, the histone SDD mutants showed reduced levels of PCNA ubiquitination after exposure to MMS or UV irradiation, consistent with a decreased MMS-induced mutagenesis relative to wild-type cells. We also found that histone SDD mutants lacking the INO80 chromatin remodeler impair HR-dependent recovery from MMS-induced replication arrest, resulting in defective S-phase progression and increased Rad52 foci. Taken together, our data provide novel insights into nucleosome functions, which link INO80-dependent chromatin remodeling to the regulation of DDT and HR during the recovery from replication blockage.

scholarly journals Interaction between DNA-damage protein GADD34 and a new member of the Hsp40 family of heat shock proteins that is induced by a DNA-damaging reagent

2000 ◽  
Vol 352 (3) ◽  
pp. 795-800 ◽  
Author(s):  
Tadao HASEGAWA ◽  
Hengyi XIAO ◽  
Fumiyasu HAMAJIMA ◽  
Ken-ichi ISOBE
Keyword(s):  
Dna Damage ◽  
Heat Shock ◽  
Cultured Cells ◽  
Similar Region ◽  
C Terminus ◽  
Mouse Tissues ◽  
Time Courses ◽  
Shock Proteins ◽  
Two Hybrid

GADD34 is one of a subset of proteins induced after DNA damage or cell growth arrest. To examine the function of GADD34, we used the yeast two-hybrid system to clone the protein that interacts with murine GADD34. As bait we used the product of the partial GADD34 cDNA, including the regions rich in proline, glutamic acid, serine and threonine (PEST) and γ134.5 regions. A cDNA clone, named GAHSP40, which is a mouse DnaJ family protein with a high similarity to human HLJ1 was cloned. The interaction between GADD34 and GAHSP40 in cultured cells was confirmed by a co-immunoprecipitation experiment and in NIH 3T3 cells by two-hybrid analysis in vivo. For binding of the two proteins, the γ134.5-similar region of GADD34 was necessary; however, the PEST region was also involved and the C-terminus of GAHSP40, but not the J-domain, was important. GAHSP40 was detected in all mouse tissues examined, but a different transcript was found in the testis. Both GADD34 mRNA and GAHSP40 mRNA were significantly elevated by treatment with methyl methanesulphonate, although the time courses were different. In addition, both GAHSP40 and GADD34 mRNA were induced by heat shock.

Sign in / Sign up

Export Citation Format

Share Document