@article{MTMT:32037907, title = {The Zn-finger of Saccharomyces cerevisiae Rad18 and its adjacent region mediate interaction with Rad5}, url = {https://m2.mtmt.hu/api/publication/32037907}, author = {Frittmann, Orsolya and Gali, Vamsi Krishna and Halmai, Miklós and Tóth, Róbert and Győrfy, Zsuzsanna and Bálint, Éva and Unk, Ildikó}, doi = {10.1093/g3journal/jkab041}, journal-iso = {G3-GENES GENOM GENET}, journal = {G3-GENES GENOMES GENETICS}, volume = {11}, unique-id = {32037907}, issn = {2160-1836}, abstract = {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.}, keywords = {Yeast two-hybrid; DNA damage tolerance; yeast genetics; Rad18-Rad5 interaction}, year = {2021}, eissn = {2160-1836}, orcid-numbers = {Gali, Vamsi Krishna/0000-0001-7048-4133} } @mastersthesis{MTMT:30611447, title = {Translesion DNA Polymerase eta functions in transcription elongation}, url = {https://m2.mtmt.hu/api/publication/30611447}, author = {Gali, Vamsi Krishna}, doi = {10.14232/phd.9838}, publisher = {SZTE}, unique-id = {30611447}, year = {2018}, orcid-numbers = {Gali, Vamsi Krishna/0000-0001-7048-4133} } @article{MTMT:3284919, title = {Translesion synthesis DNA polymerase. exhibits a specific RNA extension activity and a transcription-associated function}, url = {https://m2.mtmt.hu/api/publication/3284919}, author = {Gali, Vamsi Krishna and Bálint, Éva and Serbyn, N and Frittmann, Orsolya and Stutz, F and Unk, Ildikó}, doi = {10.1038/s41598-017-12915-1}, journal-iso = {SCI REP}, journal = {SCIENTIFIC REPORTS}, volume = {7}, unique-id = {3284919}, issn = {2045-2322}, abstract = {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.}, keywords = {IN-VITRO; ESCHERICHIA-COLI; SACCHAROMYCES-CEREVISIAE; XERODERMA-PIGMENTOSUM; Thymine dimer; POL-ETA; HUMAN NUCLEI; ELONGATION MUTANTS; RIBONUCLEOTIDE INCORPORATION; NUCLEOTIDE-EXCISION-REPAIR}, year = {2017}, eissn = {2045-2322}, orcid-numbers = {Gali, Vamsi Krishna/0000-0001-7048-4133} } @article{MTMT:3263357, title = {PCNA Retention on DNA into G2/M Phase Causes Genome Instability in Cells Lacking Elg1}, url = {https://m2.mtmt.hu/api/publication/3263357}, author = {Johnson, C and Gali, Vamsi Krishna and Takahashi, TS and Kubota, T}, doi = {10.1016/j.celrep.2016.06.030}, journal-iso = {CELL REP}, journal = {CELL REPORTS}, volume = {16}, unique-id = {3263357}, issn = {2211-1247}, abstract = {Loss of the genome maintenance factor Elg1 causes serious genome instability that leads to cancer, but the underlying mechanism is unknown. Elg1 forms the major subunit of a replication factor C-like complex, Elg1-RLC, which unloads the ring-shaped polymerase clamp PCNA from DNA during replication. Here, we show that prolonged retention of PCNA on DNA into G2/M phase is the major cause of genome instability in elg1 Delta yeast. Overexpression-induced accumulation of PCNA on DNA causes genome instability. Conversely, disassembly-prone PCNA mutants that relieve PCNA accumulation rescue the genome instability of elg1 Delta cells. Covalent modifications to the retained PCNA make only a minor contribution to elg1 Delta genome instability. By engineering cell-cycle-regulated ELG1 alleles, we show that abnormal accumulation of PCNA on DNA during S phase causes moderate genome instability and its retention through G2/M phase exacerbates genome instability. Our results reveal that PCNA unloading by Elg1-RLC is critical for genome maintenance.}, keywords = {SACCHAROMYCES-CEREVISIAE; S-PHASE; mismatch repair; NUCLEAR ANTIGEN; DAMAGE TOLERANCE; SISTER-CHROMATID COHESION; Telomere length; REPLICATION FACTOR-C; CLAMP LOADER; ALTERNATIVE RFC COMPLEX}, year = {2016}, eissn = {2211-1247}, pages = {684-695}, orcid-numbers = {Gali, Vamsi Krishna/0000-0001-7048-4133} } @article{MTMT:3123871, title = {Mutations at the Subunit Interface of Yeast Proliferating Cell Nuclear Antigen Reveal a Versatile Regulatory Domain}, url = {https://m2.mtmt.hu/api/publication/3123871}, author = {Halmai, Miklós and Frittmann, Orsolya and Szabó, Zoltán and Daraba, Andreea and Gali, Vamsi Krishna and Bálint, Éva and Unk, Ildikó}, doi = {10.1371/journal.pone.0161307}, journal-iso = {PLOS ONE}, journal = {PLOS ONE}, volume = {11}, unique-id = {3123871}, issn = {1932-6203}, abstract = {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.}, keywords = {IN-VITRO; CRYSTAL-STRUCTURE; SACCHAROMYCES-CEREVISIAE; NUCLEOTIDE EXCISION-REPAIR; TRANSLESION SYNTHESIS; AUXILIARY PROTEIN; UBIQUITIN CONJUGATION; FUNCTIONAL INTERACTIONS; REPLICATION FACTOR-C; DNA-POLYMERASE-DELTA}, year = {2016}, eissn = {1932-6203}, orcid-numbers = {Gali, Vamsi Krishna/0000-0001-7048-4133} } @article{MTMT:2524333, title = {Def1 Promotes the Degradation of Pol3 for Polymerase Exchange to Occur During DNA-Damage-Induced Mutagenesis in Saccharomyces cerevisiae.}, url = {https://m2.mtmt.hu/api/publication/2524333}, author = {Daraba, Andreea and Gali, Vamsi Krishna and Halmai, Miklós and Haracska, Lajos and Unk, Ildikó}, doi = {10.1371/journal.pbio.1001771}, journal-iso = {PLOS BIOL}, journal = {PLOS BIOLOGY}, volume = {12}, unique-id = {2524333}, issn = {1544-9173}, abstract = {DNA damages hinder the advance of replication forks because of the inability of the replicative polymerases to synthesize across most DNA lesions. Because stalled replication forks are prone to undergo DNA breakage and recombination that can lead to chromosomal rearrangements and cell death, cells possess different mechanisms to ensure the continuity of replication on damaged templates. Specialized, translesion synthesis (TLS) polymerases can take over synthesis at DNA damage sites. TLS polymerases synthesize DNA with a high error rate and are responsible for damage-induced mutagenesis, so their activity must be strictly regulated. However, the mechanism that allows their replacement of the replicative polymerase is unknown. Here, using protein complex purification and yeast genetic tools, we identify Def1 as a key factor for damage-induced mutagenesis in yeast. In in vivo experiments we demonstrate that upon DNA damage, Def1 promotes the ubiquitylation and subsequent proteasomal degradation of Pol3, the catalytic subunit of the replicative polymerase delta, whereas Pol31 and Pol32, the other two subunits of polymerase delta, are not affected. We also show that purified Pol31 and Pol32 can form a complex with the TLS polymerase Rev1. Our results imply that TLS polymerases carry out DNA lesion bypass only after the Def1-assisted removal of Pol3 from the stalled replication fork.}, year = {2014}, eissn = {1545-7885}, pages = {e1001771}, orcid-numbers = {Gali, Vamsi Krishna/0000-0001-7048-4133} }