TY - JOUR AU - Bálint, Éva AU - Unk, Ildikó TI - For the Better or for the Worse? The Effect of Manganese on the Activity of Eukaryotic DNA Polymerases JF - INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES J2 - INT J MOL SCI VL - 25 PY - 2024 IS - 1 PG - 20 SN - 1661-6596 DO - 10.3390/ijms25010363 UR - https://m2.mtmt.hu/api/publication/34547380 ID - 34547380 N1 - Funding Agency and Grant Number: ELKH Funding text: No Statement Available AB - DNA polymerases constitute a versatile group of enzymes that not only perform the essential task of genome duplication but also participate in various genome maintenance pathways, such as base and nucleotide excision repair, non-homologous end-joining, homologous recombination, and translesion synthesis. Polymerases catalyze DNA synthesis via the stepwise addition of deoxynucleoside monophosphates to the 3 ' primer end in a partially double-stranded DNA. They require divalent metal cations coordinated by active site residues of the polymerase. Mg2+ is considered the likely physiological activator because of its high cellular concentration and ability to activate DNA polymerases universally. Mn2+ can also activate the known DNA polymerases, but in most cases, it causes a significant decrease in fidelity and/or processivity. Hence, Mn2+ has been considered mutagenic and irrelevant during normal cellular function. Intriguingly, a growing body of evidence indicates that Mn2+ can positively influence some DNA polymerases by conferring translesion synthesis activity or altering the substrate specificity. Here, we review the relevant literature focusing on the impact of Mn2+ on the biochemical activity of a selected set of polymerases, namely, Pol beta, Pol lambda, and Pol mu, of the X family, as well as Pol iota and Pol eta of the Y family of polymerases, where congruous data implicate the physiological relevance of Mn2+ in the cellular function of these enzymes. LA - English DB - MTMT ER - TY - JOUR AU - Tóth, Róbert AU - Halmai, Miklós AU - Győrfy, Zsuzsanna AU - Bálint, Éva AU - Unk, Ildikó TI - The inner side of yeast PCNA contributes to genome stability by mediating interactions with Rad18 and the replicative DNA polymerase δ JF - SCIENTIFIC REPORTS J2 - SCI REP VL - 12 PY - 2022 IS - 1 SN - 2045-2322 DO - 10.1038/s41598-022-09208-7 UR - https://m2.mtmt.hu/api/publication/32762710 ID - 32762710 N1 - Funding Agency and Grant Number: ELKH Biological Research Center; National Research, Development and Innovation OfficeNational Research, Development & Innovation Office (NRDIO) - Hungary [GINOP-2.3.2-15-2016-00024] Funding text: Open access funding provided by ELKH Biological Research Center. This study was supported by National Research, Development and Innovation Office (GINOP-2.3.2-15-2016-00024). LA - English DB - MTMT ER - TY - JOUR AU - Tóth, Róbert AU - Balogh, Dávid AU - Pintér, Lajos AU - Jaksa, G. AU - Szeplaki, B. AU - Gráf, Alexandra AU - Győrfy, Zsuzsanna AU - Enyedi, Márton Zsolt AU - Kiss, Ernő AU - Haracska, Lajos AU - Unk, Ildikó TI - The Rad5 Helicase and RING Domains Contribute to Genome Stability through their Independent Catalytic Activities JF - JOURNAL OF MOLECULAR BIOLOGY J2 - J MOL BIOL VL - 434 PY - 2022 IS - 5 SN - 0022-2836 DO - 10.1016/j.jmb.2021.167437 UR - https://m2.mtmt.hu/api/publication/32606165 ID - 32606165 N1 - DNA Repair Research Group, Institute of Genetics, Biological Research Centre, Szeged, Eotvos Loránd Research Network, Szeged, H-6726, Hungary University of Szeged, Doctoral School of Biology, Hungary HCEMM-BRC Mutagenesis and Carcinogenesis Research Group, Institute of Genetics, Biological Research Centre, Szeged, Eotvos Loránd Research Network, Szeged, H-6726, Hungary Delta Bio 2000 Ltd., Szeged, H-6726, Hungary Export Date: 21 January 2022 CODEN: JMOBA Correspondence Address: Unk, I.; Biological Research Centre, Temesvari krt. 62., Hungary; email: unk.ildiko@brc.hu AB - Genomic stability is compromised by DNA damage that obstructs replication. Rad5 plays a prominent role in DNA damage bypass processes that evolved to ensure the continuation of stalled replication. Like its human orthologs, the HLTF and SHPRH tumor suppressors, yeast Rad5 has a RING domain that supports ubiquitin ligase activity promoting PCNA polyubiquitylation and a helicase domain that in the case of HLTF and Rad5 was shown to exhibit an ATPase-linked replication fork reversal activity. The RING domain is embedded in the helicase domain, confusing their separate investigation and the understanding of the exact role of Rad5 in DNA damage bypass. Particularly, it is still debated whether the helicase domain plays a catalytic or a non-enzymatic role during error-free damage bypass and whether it facilitates a function separately from the RING domain. In this study, through in vivo and in vitro characterization of domain-specific mutants, we delineate the contributions of the two domains to Rad5 function. Yeast genetic experiments and whole-genome sequencing complemented with biochemical assays demonstrate that the ubiquitin ligase and the ATPase-linked activities of Rad5 exhibit independent catalytic activities in facilitating separate pathways during error-free lesion bypass. Our results also provide important insights into the mutagenic role of Rad5 and indicate its tripartite contribution to DNA damage tolerance. © 2021 The Author(s) LA - English DB - MTMT ER - TY - JOUR AU - Bálint, Éva AU - Unk, Ildikó TI - Manganese is a strong specific activator of the rna synthetic activity of human polη JF - INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES J2 - INT J MOL SCI VL - 23 PY - 2022 IS - 1 SN - 1661-6596 DO - 10.3390/ijms23010230 UR - https://m2.mtmt.hu/api/publication/32593335 ID - 32593335 N1 - Export Date: 14 January 2022 Correspondence Address: Unk, I.; Biological Research Centre, Hungary; email: unk.ildiko@brc.hu AB - DNA polymerase η (Polη) is a translesion synthesis polymerase that can bypass different DNA lesions with varying efficiency and fidelity. Its most well-known function is the error-free bypass of ultraviolet light-induced cyclobutane pyrimidine dimers. The lack of this unique ability in humans leads to the development of a cancer-predisposing disease, the variant form of xeroderma pigmentosum. Human Polη can insert rNTPs during DNA synthesis, though with much lower efficiency than dNTPs, and it can even extend an RNA chain with ribonucleotides. We have previously shown that Mn2+ is a specific activator of the RNA synthetic activity of yeast Polη that increases the efficiency of the reaction by several thousand-fold over Mg2+. In this study, our goal was to investi-gate the metal cofactor dependence of RNA synthesis by human Polη. We found that out of the investigated metal cations, only Mn2+ supported robust RNA synthesis. Steady state kinetic analysis showed that Mn2+ activated the reaction a thousand-fold compared to Mg2+, even during DNA damage bypass opposite 8-oxoG and TT dimer. Our results revealed a two order of magnitude higher affinity of human Polη towards ribonucleotides in the presence of Mn2+ compared to Mg2+. It is note-worthy that activation occurred without lowering the base selectivity of the enzyme on undamaged templates, whereas the fidelity decreased across a TT dimer. In summary, our data strongly suggest that, like with its yeast homolog, Mn2+ is the proper metal cofactor of hPolη during RNA chain extension, and selective metal cofactor utilization contributes to switching between its DNA and RNA synthetic activities. © 2021 by the authors. Licensee MDPI, Basel, Switzerland. LA - English DB - MTMT ER - TY - JOUR AU - Frittmann, Orsolya AU - Gali, Vamsi Krishna AU - Halmai, Miklós AU - Tóth, Róbert AU - Győrfy, Zsuzsanna AU - Bálint, Éva AU - Unk, Ildikó TI - The Zn-finger of Saccharomyces cerevisiae Rad18 and its adjacent region mediate interaction with Rad5 JF - G3-GENES GENOMES GENETICS J2 - G3-GENES GENOM GENET VL - 11 PY - 2021 IS - 4 PG - 12 SN - 2160-1836 DO - 10.1093/g3journal/jkab041 UR - https://m2.mtmt.hu/api/publication/32037907 ID - 32037907 N1 - Biological Research Centre, Eotvos Loránd Research Network, Institute of Genetics, Szeged, H-6726, Hungary Doctoral School of Biology, University of Szeged, Szeged, H-6720, Hungary Export Date: 27 May 2021 Biological Research Centre, Institute of Genetics, Szeged, H-6726, Hungary Doctoral School of Biology, University of Szeged, Szeged, H-6720, Hungary Export Date: 31 August 2021 Correspondence Address: Unk, I.; Biological Research Centre, Temesvari krt. 62, Hungary; email: unk.ildiko@brc.hu AB - DNA damages that hinder the movement of the replication complex can ultimately lead to cell death. To avoid that, cells possess several DNA damage bypass mechanisms. The Rad18 ubiquitin ligase controls error-free and mutagenic pathways that help the replication complex to bypass DNA lesions by monoubiquitylating PCNA at stalled replication forks. In Saccharomyces cerevisiae, two of the Rad18 governed pathways are activated by monoubiquitylated PCNA and they involve translesion synthesis polymerases, whereas a third pathway needs subsequent polyubiquitylation of the same PCNA residue by another ubiquitin ligase the Rad5 protein, and it employs template switching. The goal of this study was to dissect the regulatory role of the multidomain Rad18 in DNA damage bypass using a structure-function based approach. Investigating deletion and point mutant RAD18 variants in yeast genetic and yeast two-hybrid assays we show that the Zn-finger of Rad18 mediates its interaction with Rad5, and the N-terminal adjacent region is also necessary for Rad5 binding. Moreover, results of the yeast two-hybrid and in vivo ubiquitylation experiments raise the possibility that direct interaction between Rad18 and Rad5 might not be necessary for the function of the Rad5 dependent pathway. The presented data also reveal that yeast Rad18 uses different domains to mediate its association with itself and with Rad5. Our results contribute to better understanding of the complex machinery of DNA damage bypass pathways. © The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America. LA - English DB - MTMT ER - TY - JOUR AU - Bálint, Éva AU - Unk, Ildikó TI - Selective Metal Ion Utilization Contributes to the Transformation of the Activity of Yeast Polymerase eta from DNA Polymerization toward RNA Polymerization JF - INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES J2 - INT J MOL SCI VL - 21 PY - 2020 IS - 21 SN - 1661-6596 DO - 10.3390/ijms21218248 UR - https://m2.mtmt.hu/api/publication/31682228 ID - 31682228 AB - Polymerase eta (Pol eta) is a translesion synthesis DNA polymerase directly linked to cancer development. It can bypass several DNA lesions thereby rescuing DNA damage-stalled replication complexes. We previously presented evidence implicating Saccharomyces cerevisiae Pol eta in transcription elongation, and identified its specific RNA extension and translesion RNA synthetic activities. However, RNA synthesis by Pol eta proved rather inefficient under conditions optimal for DNA synthesis. Searching for factors that could enhance its RNA synthetic activity, we have identified the divalent cation of manganese. Here, we show that manganese triggers drastic changes in the activity of Pol eta. Kinetics experiments indicate that manganese increases the efficiency of ribonucleoside incorporation into RNA by similar to 400-2000-fold opposite undamaged DNA, and similar to 3000 and similar to 6000-fold opposite TT dimer and 8oxoG, respectively. Importantly, preference for the correct base is maintained with manganese during RNA synthesis. In contrast, activity is strongly impaired, and base discrimination is almost lost during DNA synthesis by Pol eta with manganese. Moreover, Pol eta shows strong preference for manganese during RNA synthesis even at a 25-fold excess magnesium concentration. Based on this, we suggest that a new regulatory mechanism, selective metal cofactor utilization, modulates the specificity of Pol eta helping it to perform distinct activities needed for its separate functions during replication and transcription. LA - English DB - MTMT ER - TY - JOUR AU - Gali, Vamsi Krishna AU - Bálint, Éva AU - Serbyn, N AU - Frittmann, Orsolya AU - Stutz, F AU - Unk, Ildikó TI - Translesion synthesis DNA polymerase. exhibits a specific RNA extension activity and a transcription-associated function JF - SCIENTIFIC REPORTS J2 - SCI REP VL - 7 PY - 2017 IS - 1 PG - 17 SN - 2045-2322 DO - 10.1038/s41598-017-12915-1 UR - https://m2.mtmt.hu/api/publication/3284919 ID - 3284919 N1 - OA gold \n Institute of Genetics, Biological Research Centre, Hungarian Academy of Sciences, Szeged, H-6726, Hungary \n Department of Cell Biology, IGE3, University of Geneva, Geneva, 1211, Switzerland \n Institute of Medical Sciences Foresterhill, University of Aberdeen, Aberdeen, United Kingdom \n Cited By :2 \n Export Date: 30 November 2018 \n Correspondence Address: Unk, I.; Institute of Genetics, Biological Research Centre, Hungarian Academy of SciencesHungary; email: unk.ildiko@brc.mta.hu AB - Polymerase eta (Pol eta) is a low fidelity translesion synthesis DNA polymerase that rescues damage-stalled replication by inserting deoxy-ribonucleotides opposite DNA damage sites resulting in error-free or mutagenic damage bypass. In this study we identify a new specific RNA extension activity of Pol eta of Saccharomyces cerevisiae. We show that Pol eta is able to extend RNA primers in the presence of ribonucleotides (rNTPs), and that these reactions are an order of magnitude more efficient than the misinsertion of rNTPs into DNA. Moreover, during RNA extension Pol eta performs error-free bypass of the 8-oxoguanine and thymine dimer DNA lesions, though with a 10(3) and 10(2)-fold lower efficiency, respectively, than it synthesizes opposite undamaged nucleotides. Furthermore, in vivo experiments demonstrate that the transcription of several genes is affected by the lack of Pol eta, and that Pol eta is enriched over actively transcribed regions. Moreover, inactivation of its polymerase activity causes similar transcription inhibition as the absence of Pol eta. In summary, these results suggest that the new RNA synthetic activity of Pol eta can have in vivo relevance. LA - English DB - MTMT ER - TY - JOUR AU - Halmai, Miklós AU - Frittmann, Orsolya AU - Szabó, Zoltán AU - Daraba, Andreea AU - Gali, Vamsi Krishna AU - Bálint, Éva AU - Unk, Ildikó TI - Mutations at the Subunit Interface of Yeast Proliferating Cell Nuclear Antigen Reveal a Versatile Regulatory Domain JF - PLOS ONE J2 - PLOS ONE VL - 11 PY - 2016 IS - 8 PG - 20 SN - 1932-6203 DO - 10.1371/journal.pone.0161307 UR - https://m2.mtmt.hu/api/publication/3123871 ID - 3123871 N1 - Cited By :4 Export Date: 11 July 2022 CODEN: POLNC AB - Proliferating cell nuclear antigen (PCNA) plays a key role in many cellular processes and due to that it interacts with a plethora of proteins. The main interacting surfaces of Saccharomyces cerevisiae PCNA have been mapped to the interdomain connecting loop and to the carboxy-terminal domain. Here we report that the subunit interface of yeast PCNA also has regulatory roles in the function of several DNA damage response pathways. Using sitedirected mutagenesis we engineered mutations at both sides of the interface and investigated the effect of these alleles on DNA damage response. Genetic experiments with strains bearing the mutant alleles revealed that mutagenic translesion synthesis, nucleotide excision repair, and homologous recombination are all regulated through residues at the subunit interface. Moreover, genetic characterization of one of our mutants identifies a new sub-branch of nucleotide excision repair. Based on these results we conclude that residues at the subunit boundary of PCNA are not only important for the formation of the trimer structure of PCNA, but they constitute a regulatory protein domain that mediates different DNA damage response pathways, as well. LA - English DB - MTMT ER - TY - JOUR AU - Unk, Ildikó TI - DNS-javítás. A 2015-ös kémiai Nobel-díj JF - TERMÉSZET VILÁGA J2 - TERMÉSZET VILÁGA VL - 147 PY - 2016 IS - 2 SP - 50 EP - 54 PG - 5 SN - 0040-3717 UR - https://m2.mtmt.hu/api/publication/3022934 ID - 3022934 LA - Hungarian DB - MTMT ER - TY - JOUR AU - Unk, Ildikó AU - Daraba, Andreea TI - Measuring UV-induced Mutagenesis at the CAN1 Locus in Saccharomyces cerevisiae. JF - BIO-PROTOCOL J2 - BIO-PROTOCOL VL - 4 PY - 2014 IS - 20 PG - 6 SN - 2331-8325 DO - 10.21769/BioProtoc.1272 UR - https://m2.mtmt.hu/api/publication/3287841 ID - 3287841 AB - There are several methods to measure the capacity of yeast cell to respond to environmental impacts on their genome by mutating it. One frequently used method involves the detection of forward mutations in the CAN1 gene. The CAN1 gene encodes for an arginine permease that is responsible for the uptake of arginine and it can also transport the toxic analog of arginine, canavanine (Whelan et al., 1979). When CAN1 cells are grown on a media containing canavanine but lacking arginine, the cells die because of the uptake of the toxic canavanine. However, if a mutation in the CAN1 gene inactivates the permease, that cell survives and forms a colony on the plate. The following protocol describes the measurement of UV-induced mutagenesis at the CAN1 locus. LA - English DB - MTMT ER -