The Experts below are selected from a list of 924 Experts worldwide ranked by ideXlab platform
Fumio Hanaoka - One of the best experts on this subject based on the ideXlab platform.
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structure and mechanism of human DNA Polymerase η
Nature, 2010Co-Authors: Christian Biertumpfel, Chikahide Masutani, Alan R Lehmann, Ye Zhao, Yuji Kondo, Santiago Ramonmaiques, Mark S Gregory, Fumio HanaokaAbstract:The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA Polymerase Eta (Pol Eta), a DNA Polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Pol Eta at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Pol Eta acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-mEtal ion catalysis. Two residues conserved among Pol Eta orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. On the basis of the structures, eight Pol Eta missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Pol Eta in replicating through D loop and DNA fragile sites.
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The Molecular Chaperone Hsp90 Regulates Accumulation of DNA Polymerase η at Replication Stalling Sites in UV-Irradiated Cells
Molecular cell, 2010Co-Authors: Takayuki Sekimoto, Fumio Hanaoka, Chikahide Masutani, Tsukasa Oda, Franklin Mayca Pozo, Yoshiki Murakumo, Takayuki YamashitaAbstract:DNA Polymerase Eta (Pol Eta) is a member of the mammalian Y family Polymerases and performs error-free translesion synthesis across UV-damaged DNA. For this function, Pol Eta accumulates in nuclear foci at replication stalling sites via its interaction with monoubiquitinated PCNA. However, little is known about the posttranslational control mechanisms of Pol Eta, which regulate its accumulation in replication foci. Here, we report that the molecular chaperone Hsp90 promotes UV irradiation-induced nuclear focus formation of Pol Eta through control of its stability and binding to monoubiquitinated PCNA. Our data indicate that Hsp90 facilitates the folding of Pol Eta into an active form in which PCNA- and ubiquitin-binding regions are functional. Furthermore, Hsp90 inhibition potentiates UV-induced cytotoxicity and mutagenesis in a Pol Eta-dependent manner. Our studies identify Hsp90 as an essential regulator of Pol Eta-mediated translesion synthesis.
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Characterization of a Y-Family DNA Polymerase Eta from the Eukaryotic Thermophile Alvinella pompejana
Hindawi Limited, 2010Co-Authors: Sayo Kashiwagi, Chikahide Masutani, Isao Kuraoka, Yoshie Fujiwara, Kenichi Hitomi, Quen J. Cheng, Jill O. Fuss, David S. Shin, John A. Tainer, Fumio HanaokaAbstract:Human DNA Polymerase η (HsPolη) plays an important role in translesion synthesis (TLS), which allows for replication past DNA damage such as UV-induced cis-syn cyclobutane pyrimidine dimers (CPDs). Here, we characterized ApPolη from the thermophilic worm Alvinella pompejana, which inhabits deep-sea hydrothermal vent chimneys. ApPolη shares sequence homology with HsPolη and contains domains for binding ubiquitin and proliferating cell nuclear antigen. Sun-induced UV does not penetrate Alvinella's environment; however, this novel DNA Polymerase catalyzed efficient and accurate TLS past CPD, as well as 7,8-dihydro-8-oxoguanine and isomers of thymine glycol induced by reactive oxygen species. In addition, we found that ApPolη is more thermostable than HsPolη, as expected from its habitat temperature. Moreover, the activity of this enzyme was rEtained in the presence of a higher concentration of organic solvents. Therefore, ApPolη provides a robust, human-like Polη that is more active after exposure to high temperatures and organic solvents
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Rad54 dissociates homologous recombination intermediates by branch migration
Nature structural & molecular biology, 2007Co-Authors: Dmitry V. Bugreev, Fumio Hanaoka, Alexander V. MazinAbstract:Double-strand DNA breaks (DSBs) cause cell death and genome instability. Homologous recombination is a major DSB repair pathway that operates by forming joint molecules with homologous DNA sequences, which are used as templates to achieve accurate repair. In eukaryotes, Rad51 protein (RecA homolog) searches for homologous sequences and catalyzes the formation of joint molecules (D-loops). Once joint molecules have been formed, DNA Polymerase extends the 3' single-stranded DNA tails of the broken chromosome, restoring the lost information. How joint molecules subsequently dissociate is unknown. We reconstituted DSB repair in vitro using purified human homologous recombination proteins and DNA Polymerase Eta. We found that Rad54 protein, owing to its ATP-dependent branch-migration activity, can cause dissociation of joint molecules. These results suggest a previously uncharacterized mechanism of DSB repair in which Rad54 branch-migration activity plays an important role.
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2 hydroxy 2 deoxyadenosine 5 triphosphate enhances a t c g mutations caused by 8 hydroxy 2 deoxyguanosine 5 triphosphate by suppressing its degradation upon replication in a hela extract
Biochemistry, 2007Co-Authors: Kazuya Satou, Fumio Hanaoka, Hideyoshi Harashima, Chikahide Masutani, Hiroshi Kasai, Hiroyuki KamiyaAbstract:The coexistence effects of multiple kinds of oxidized deoxyribonucleotides were examined using an SV40 origin-dependent in vitro replication system with a HeLa extract. Oxidized dGTP and dATP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP) and 2-hydroxy-2'-deoxyadenosine 5'-triphosphate (2-OH-dATP), were used in this study. The mutation frequency synergistically increased when the two oxidized deoxyribonucleotides were together in the reaction. 2-OH-dATP enhanced the mutagenicity of 8-OH-dGTP, since the induced mutations were A.T --> C.G transversions. The contribution of the highly error-prone DNA Polymerase Eta was unlikely, since similar results were observed with an XP-V cell extract. The possible involvement of 2-hydroxyadenine in the complementary (template) strand was excluded on the basis of experiments using plasmids containing 2-hydroxyadenine as templates in the reactions with 8-OH-dGTP. 2-OH-dATP suppressed hydrolysis of 8-OH-dGTP, suggesting that the inhibition of the MTH1 protein played the major role in the enhancement. These results highlight the importance of specific hydrolysis of 8-OH-dGTP for the suppression of its induced mutation.
Alan R Lehmann - One of the best experts on this subject based on the ideXlab platform.
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structure and mechanism of human DNA Polymerase η
Nature, 2010Co-Authors: Christian Biertumpfel, Chikahide Masutani, Alan R Lehmann, Ye Zhao, Yuji Kondo, Santiago Ramonmaiques, Mark S Gregory, Fumio HanaokaAbstract:The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA Polymerase Eta (Pol Eta), a DNA Polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Pol Eta at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Pol Eta acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-mEtal ion catalysis. Two residues conserved among Pol Eta orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. On the basis of the structures, eight Pol Eta missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Pol Eta in replicating through D loop and DNA fragile sites.
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Regulation of Translesion Synthesis DNA Polymerase η by Monoubiquitination
Molecular cell, 2010Co-Authors: Marzena Bienko, Catherine M. Green, Simone Sabbioneda, Nicola Crosetto, Ivan Matic, Richard G. Hibbert, Tihana Begovic, Atsuko Niimi, Matthias Mann, Alan R LehmannAbstract:DNA Polymerase Eta is a Y family Polymerase involved in translesion synthesis (TLS). Its action is initiated by simultaneous interaction between the PIP box in pol Eta and PCNA and between the UBZ in pol Eta and monoubiquitin attached to PCNA. Whereas monoubiquitination of PCNA is required for its interaction with pol Eta during TLS, we now show that monoubiquitination of pol Eta inhibits this interaction, preventing its functions in undamaged cells. Identification of monoubiquitination sites within pol Eta nuclear localization signal (NLS) led to the discovery that pol Eta NLS directly contacts PCNA, forming an extended pol Eta-PCNA interaction surface. We name this the PCNA-interacting region (PIR) and show that its monoubiquitination is downregulated by various DNA-damaging agents. We propose that this mechanism ensures optimal availability of nonubiquitinated, TLS-competent pol Eta after DNA damage. Our work shows how monoubiquitination can either positively or negatively regulate the assembly of a protein complex, depending on which substrates are targeted by ubiquitin.
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Localization of Y-family Polymerases and the DNA Polymerase switch in mammalian cells.
Methods in enzymology, 2006Co-Authors: Patricia Kannouche, Alan R LehmannAbstract:During translesion synthesis past sites of damaged DNA, specialized Y-family Polymerases are employed by the cell to replace the high stringency replicative Polymerases and synthesize DNA past the damaged site. These Polymerases are localized in replication factories during the S phase of the cell cycle. When progress of the replication fork is blocked, the Polymerase accessory protein, proliferating cell nuclear antigen (PCNA), becomes ubiquitinated and the monoubiquitinated PCNA has an increased affinity for Y-family DNA Polymerase Eta (polEta). This chapter describes methods for visualizing the Polymerases in replication factories, for analyzing the ubiquitination status of PCNA, and for measuring its interaction with polEta in chromatin extracts.
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interaction of human DNA Polymerase η with monoubiquitinated pcna a possible mechanism for the Polymerase switch in response to DNA damage
Molecular Cell, 2004Co-Authors: Patricia Kannouche, Jonathan F Wing, Alan R LehmannAbstract:Most types of DNA damage block replication fork progression during DNA synthesis because replicative DNA Polymerases are unable to accommodate altered DNA bases in their active sites. To overcome this block, eukaryotic cells employ specialized translesion synthesis (TLS) Polymerases, which can insert nucleotides opposite damaged bases. In particular, TLS by DNA Polymerase Eta (polEta) is the major pathway for bypassing UV photoproducts. How the cell switches from replicative to TLS Polymerase at the site of blocked forks is unknown. We show that, in human cells, PCNA becomes monoubiquitinated following UV irradiation of the cells and that this is dependent on the hRad18 protein. Monoubiquitinated PCNA but not unmodified PCNA specifically interacts with polEta, and we have identified two motifs in polEta that are involved in this interaction. Our findings provide an attractive mechanism by which monoubiquitination of PCNA might mediate the Polymerase switch.
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Replication of damaged DNA in mammalian cells: new solutions to an old problem.
Mutation research, 2002Co-Authors: Alan R LehmannAbstract:All cells need not only to remove damage from their DNA, but also to be able to replicate DNA containing unrepaired damage. In mammalian cells, the major process by which cells are able to replicate damaged templates is translesion synthesis, the direct synthesis of DNA past altered bases. Crucial to this process is a series of recently discovered DNA Polymerases. Most of them belong to a new family of Polymerases designated the Y-family, which have conserved sequences in the catalytic N-terminal half of the proteins. These Polymerases have different efficiencies and specificities in vitro depending on the type of damage in the template.One of them, DNA Polymerase Eta, is defective in xeroderma pigmentosum variants, and overwhelming evidence suggests that this is the Polymerase that carries out translesion synthesis past UV-induced cyclobutane pyrimidine dimers in vivo. DNA Polymerase Eta is localised in replication factories during DNA replication and accumulates at sites of stalled replication forks. Many studies have been carried out on the properties of the other Polymerases in vitro, but there is as yet very little evidence for their specific roles in vivo.
Altaf A. Wani - One of the best experts on this subject based on the ideXlab platform.
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USP7 modulates UV-induced PCNA monoubiquitination by regulating DNA Polymerase Eta stability
Oncogene, 2014Co-Authors: Jiang Qian, Kyle Pentz, Qi-en Wang, Qianzheng Zhu, Amit Kumar Srivastava, Altaf A. WaniAbstract:DNA Polymerase Eta (Polη) has unique and pivotal functions in several DNA damage-tolerance pathways. Steady-state level of this short-lived protein is tightly controlled by multiple mechanisms including proteolysis. Here, we have identified the deubiquitinating enzyme (DUB), ubiquitin-specific protease 7 (USP7), as a novel regulator of Polη stability. USP7 regulates Polη stability through both indirect and direct mechanisms. Knockout of USP7 increased the steady-state level of Polη and slowed down the turnover of both Polη and p53 proteins through destabilizing their E3 ligase murine double minute 2 (Mdm2). Also, USP7 physically binds Polη in vitro and in vivo. Overexpression of wild-type USP7 but not its catalytically-defective mutants deubiquitinates Polη and increases its cellular steady-state level. Thus, USP7 directly serves as a specific DUB for Polη. Furthermore, ectopic expression of USP7 promoted the UV-induced proliferating cell nuclear antigen (PCNA) monoubiquitination in Polη-proficient but not in Polη-deficient XPV (Xeroderma pigmentosum variant) cells, suggesting that USP7 facilitates UV-induced PCNA monoubiquitination by stabilizing Polη. Taken together, our findings reveal a modulatory role of USP7 in PCNA ubiquitination-mediated stress-tolerance pathways by fine-tuning Polη turnover.
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Abstract 4893: USP7 modulates UV-induced PCNA monoubiquitylation by regulating DNA Polymerase Eta stability
Tumor Biology, 2014Co-Authors: Jiang Qian, Qianzhen Zhu, Kyle Pentz, Qi-en Wang, Altaf A. WaniAbstract:Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA DNA Polymerase Eta (Polη), a product of the Xeroderma pigmentosum variant (XPV) gene, plays a unique role in translesion DNA synthesis (TLS). It is a short-lived proteasomally degraded protein and its steady-state level is tightly controlled by multiple pathways. In this study, we have identified the deubiquitylating enzyme ubiquitin-specific protease 7 (USP7) as a novel regulator of Polη stability. Polη and USP7 interact in vitro and in vivo. Overexpression of wild-type USP7, but not its interaction-defective and catalytic mutants, deubiquitylate Polη and increase its steady-state level. The data demonstrate that both physical interaction and catalytic activity of USP7 are necessary for Polη deubiquitylation and stability regulation. Interestingly, knockout of USP7 also increased the steady-state levels and slowed the turnover of both Polη and p53, which is likely caused by destabilizing MDM2, a targeting E3 ligase for ubiquitylation of both Polη and p53. Furthermore, ectopic expression of USP7 upregulated the UV-induced PCNA monoubiquitylation. In contrast, UV-induced PCNA monoubiquitylation was not observed in XPV cells ectopically expressing USP7. Taken together, our results demonstrated that USP7 facilitates UV-induced PCNA monoubiquitylation by directly and indirectly regulating Polη stability. (The work was supported by grants from NIH.) Note: This abstract was not presented at the meeting. Citation Format: Jiang Qian, Qianzhen Zhu, Kyle Pentz, Jinshan He, Qien Wang, Altaf A. Wani. USP7 modulates UV-induced PCNA monoubiquitylation by regulating DNA Polymerase Eta stability. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4893. doi:10.1158/1538-7445.AM2014-4893
Chikahide Masutani - One of the best experts on this subject based on the ideXlab platform.
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structure and mechanism of human DNA Polymerase η
Nature, 2010Co-Authors: Christian Biertumpfel, Chikahide Masutani, Alan R Lehmann, Ye Zhao, Yuji Kondo, Santiago Ramonmaiques, Mark S Gregory, Fumio HanaokaAbstract:The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA Polymerase Eta (Pol Eta), a DNA Polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Pol Eta at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Pol Eta acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-mEtal ion catalysis. Two residues conserved among Pol Eta orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. On the basis of the structures, eight Pol Eta missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Pol Eta in replicating through D loop and DNA fragile sites.
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The Molecular Chaperone Hsp90 Regulates Accumulation of DNA Polymerase η at Replication Stalling Sites in UV-Irradiated Cells
Molecular cell, 2010Co-Authors: Takayuki Sekimoto, Fumio Hanaoka, Chikahide Masutani, Tsukasa Oda, Franklin Mayca Pozo, Yoshiki Murakumo, Takayuki YamashitaAbstract:DNA Polymerase Eta (Pol Eta) is a member of the mammalian Y family Polymerases and performs error-free translesion synthesis across UV-damaged DNA. For this function, Pol Eta accumulates in nuclear foci at replication stalling sites via its interaction with monoubiquitinated PCNA. However, little is known about the posttranslational control mechanisms of Pol Eta, which regulate its accumulation in replication foci. Here, we report that the molecular chaperone Hsp90 promotes UV irradiation-induced nuclear focus formation of Pol Eta through control of its stability and binding to monoubiquitinated PCNA. Our data indicate that Hsp90 facilitates the folding of Pol Eta into an active form in which PCNA- and ubiquitin-binding regions are functional. Furthermore, Hsp90 inhibition potentiates UV-induced cytotoxicity and mutagenesis in a Pol Eta-dependent manner. Our studies identify Hsp90 as an essential regulator of Pol Eta-mediated translesion synthesis.
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Characterization of a Y-Family DNA Polymerase Eta from the Eukaryotic Thermophile Alvinella pompejana
Hindawi Limited, 2010Co-Authors: Sayo Kashiwagi, Chikahide Masutani, Isao Kuraoka, Yoshie Fujiwara, Kenichi Hitomi, Quen J. Cheng, Jill O. Fuss, David S. Shin, John A. Tainer, Fumio HanaokaAbstract:Human DNA Polymerase η (HsPolη) plays an important role in translesion synthesis (TLS), which allows for replication past DNA damage such as UV-induced cis-syn cyclobutane pyrimidine dimers (CPDs). Here, we characterized ApPolη from the thermophilic worm Alvinella pompejana, which inhabits deep-sea hydrothermal vent chimneys. ApPolη shares sequence homology with HsPolη and contains domains for binding ubiquitin and proliferating cell nuclear antigen. Sun-induced UV does not penetrate Alvinella's environment; however, this novel DNA Polymerase catalyzed efficient and accurate TLS past CPD, as well as 7,8-dihydro-8-oxoguanine and isomers of thymine glycol induced by reactive oxygen species. In addition, we found that ApPolη is more thermostable than HsPolη, as expected from its habitat temperature. Moreover, the activity of this enzyme was rEtained in the presence of a higher concentration of organic solvents. Therefore, ApPolη provides a robust, human-like Polη that is more active after exposure to high temperatures and organic solvents
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2 hydroxy 2 deoxyadenosine 5 triphosphate enhances a t c g mutations caused by 8 hydroxy 2 deoxyguanosine 5 triphosphate by suppressing its degradation upon replication in a hela extract
Biochemistry, 2007Co-Authors: Kazuya Satou, Fumio Hanaoka, Hideyoshi Harashima, Chikahide Masutani, Hiroshi Kasai, Hiroyuki KamiyaAbstract:The coexistence effects of multiple kinds of oxidized deoxyribonucleotides were examined using an SV40 origin-dependent in vitro replication system with a HeLa extract. Oxidized dGTP and dATP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP) and 2-hydroxy-2'-deoxyadenosine 5'-triphosphate (2-OH-dATP), were used in this study. The mutation frequency synergistically increased when the two oxidized deoxyribonucleotides were together in the reaction. 2-OH-dATP enhanced the mutagenicity of 8-OH-dGTP, since the induced mutations were A.T --> C.G transversions. The contribution of the highly error-prone DNA Polymerase Eta was unlikely, since similar results were observed with an XP-V cell extract. The possible involvement of 2-hydroxyadenine in the complementary (template) strand was excluded on the basis of experiments using plasmids containing 2-hydroxyadenine as templates in the reactions with 8-OH-dGTP. 2-OH-dATP suppressed hydrolysis of 8-OH-dGTP, suggesting that the inhibition of the MTH1 protein played the major role in the enhancement. These results highlight the importance of specific hydrolysis of 8-OH-dGTP for the suppression of its induced mutation.
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Efficient and Erroneous Incorporation of Oxidized DNA Precursors by Human DNA Polymerase η
Biochemistry, 2007Co-Authors: Masatomi Shimizu, Fumio Hanaoka, Hiroyuki Kamiya, Hideyoshi Harashima, Hiroshi Sugiyama, Chikahide Masutani, Petr Grúz, Yukio Usui, Takehiko NohmiAbstract:Altered oxidative mEtabolism is a property of many tumor cells. Oxidation of DNA precursors, i.e., dNTP pool, as well as DNA is a major source of mutagenesis and carcinogenesis. Here, we report the remarkable nature of human DNA Polymerase Eta that incorporates oxidized dNTPs into a nascent DNA strand in an efficient and erroneous manner. The Polymerase almost exclusively incorporated 8-hydroxy-dGTP (8-OH-dGTP) opposite template adenine (A) at 60% efficiency of normal dTTP incorporation, and incorporated 2-hydroxy-dATP (2-OH-dATP) opposite template thymine (T), guanine (G), or cytosine (C) at substantial rates. The synthetic primers having 8-hydroxy-G paired with template A or 2-hydroxy-A paired with template T, G, or C at the termini were efficiently extended. In contrast, human DNA Polymerase iota incorporated 8-OH-dGTP opposite template A with much lower efficiency and did not incorporate 2-OH-dATP opposite any of the template bases. It did not extend the primers having the oxidized bases at the termini either. We propose that human DNA Polymerase Eta may participate in oxidative mutagenesis through the efficient and erroneous incorporation of oxidized dNTPs during DNA synthesis.
Narottam Acharya - One of the best experts on this subject based on the ideXlab platform.
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Multifaceted activities of DNA Polymerase η: beyond translesion DNA synthesis
Current Genetics, 2019Co-Authors: Narottam Acharya, Kodavati Manohar, Doureradjou Peroumal, Prashant Khandagale, Shraddheya Kumar Patel, Satya Ranjan Sahu, Premlata KumariAbstract:DNA Polymerases are evolved to extend the 3′-OH of a growing primer annealed to a template DNA substrate. Since replicative DNA Polymerases have a limited role while replicating structurally distorted template, translesion DNA Polymerases mostly from Y-family come to the rescue of stalled replication fork and maintain genome stability. DNA Polymerase Eta is one such specialized enzyme whose function is directly associated with casual development of certain skin cancers and chemo-resistance. More than 20 years of extensive studies are available to support TLS activities of Polη in bypassing various DNA lesions, in addition, limited but crucial growing evidence also exist to suggest Polη possessing TLS-independent cellular functions. In this review, we have mostly focused on non-TLS activities of Polη from different organisms including our recent findings from pathogenic yeast Candida albicans .
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tls dependent and independent functions of DNA Polymerase Eta polη rad30 from pathogenic yeast candida albicans
Molecular Microbiology, 2018Co-Authors: Kodavati Manohar, Doureradjou Peroumal, Narottam AcharyaAbstract:Polη, a unique TLS DNA Polymerase that promotes efficient bypass of UV-induced CPDs and cisplatin adducts, has not been explored in Candida species yet. Here, we show that CaPolη plays a vital role in protecting Candida albicans genome from diverse array of DNA damaging agents, not limited to UV and cisplatin. Polη deficient strain did not exhibit any hyphal development in the presence of UV and cisplatin while the wild type strain profusely developed DNA damage induced filamentation. The polarized growth induced by HU and MMS was found to be Polη independent. No common regulatory pathway of morphogenesis operates in C. albicans due to genomic stress, rather Polη branches away from RAD53 dependent pathway to be specific to UV/cisplatin. Interestingly, serum that does not inflict any DNA damage also induces hyphal growth in C. albicans, and requires a functionally active Polη. Importantly, deletion of RAD30 sensitized the strain to amphotericin B; but its presence resulted in azole drug tolerance only in DNA damaging conditions. We suggest that the roles of CaPolη in genome stability and genotoxins induced filamentation are due to its TLS activities; whereas its TLS independent functions play a vital role in serum induced morphogenesis and amphotericin B resistance.
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TLS dependent and independent functions of DNA Polymerase Eta (Polη/Rad30) from Pathogenic Yeast Candida albicans.
Molecular microbiology, 2018Co-Authors: Kodavati Manohar, Doureradjou Peroumal, Narottam AcharyaAbstract:Polη, a unique TLS DNA Polymerase that promotes efficient bypass of UV-induced CPDs and cisplatin adducts, has not been explored in Candida species yet. Here, we show that CaPolη plays a vital role in protecting Candida albicans genome from diverse array of DNA damaging agents, not limited to UV and cisplatin. Polη deficient strain did not exhibit any hyphal development in the presence of UV and cisplatin while the wild type strain profusely developed DNA damage induced filamentation. The polarized growth induced by HU and MMS was found to be Polη independent. No common regulatory pathway of morphogenesis operates in C. albicans due to genomic stress, rather Polη branches away from RAD53 dependent pathway to be specific to UV/cisplatin. Interestingly, serum that does not inflict any DNA damage also induces hyphal growth in C. albicans, and requires a functionally active Polη. Importantly, deletion of RAD30 sensitized the strain to amphotericin B; but its presence resulted in azole drug tolerance only in DNA damaging conditions. We suggest that the roles of CaPolη in genome stability and genotoxins induced filamentation are due to its TLS activities; whereas its TLS independent functions play a vital role in serum induced morphogenesis and amphotericin B resistance.
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Comparative molecular dynamics studies of heterozygous open reading frames of DNA Polymerase Eta (η) in pathogenic yeast Candida albicans
Scientific Reports, 2017Co-Authors: Suresh Satpati, Kodavati Manohar, Narottam Acharya, Anshuman DixitAbstract:Genomic instability in Candida albicans is believed to play a crucial role in fungal pathogenesis. DNA Polymerases contribute significantly to stability of any genome. Although Candida Genome database predicts presence of S. cerevisiae DNA Polymerase orthologs; functional and structural characterizations of Candida DNA Polymerases are still unexplored. DNA Polymerase Eta (Polη) is unique as it promotes efficient bypass of cyclobutane pyrimidine dimers. Interestingly, C. albicans is heterozygous in carrying two Polη genes and the nucleotide substitutions were found only in the ORFs. As allelic differences often result in functional differences of the encoded proteins, comparative analyses of structural models and molecular dynamic simulations were performed to characterize these orthologs of DNA Polη. Overall structures of both the ORFs remain conserved except subtle differences in the palm and PAD domains. The complementation analysis showed that both the ORFs equally suppressed UV sensitivity of yeast rad30 deletion strain. Our study has predicted two novel molecular interactions, a highly conserved molecular tetrad of salt bridges and a series of π–π interactions spanning from thumb to PAD. This study suggests these ORFs as the homologues of yeast Polη, and due to its heterogeneity in C. albicans they may play a significant role in pathogenicity.