Az UV-sugárzás által károsított DNS Rad6 ubiquitin-konjugáló enzim által irányított, mutációt okozó illetve hibamentes replikációja Saccharomyces cerevisiae-ben = The Rad6 ubiquitin-conjugating enzyme dependent error-free and error-prone translesion DNA synthesis of UV-damaged DNA in Saccharomyces cerevisiae

A környezetünkben jelenlévő és a metabolikusan keletkező reaktív ágensek is folyamatosan károsítják a DNS-t. A kijavítatlan DNS hibáknál elakadt replikációs villa mentésében játszik szerepet a RAD6-RAD18-függő DNS károsodást toleráló mechanizmus, mely hibamentesen vagy mutációt generálva vezethet a károsodott DNS replikációjához. Kutatásunk célja az volt, hogy mélyebb betekintést nyerjünk a Saccharomyces cerevisiae RAD6, RAD18, RAD5, PCNA, UBC13, MMS2, REV7, és REV1 gének szerepére a károsított DNS replikációjában. Fehérje-komplexek kettős affinitás tisztításával és élesztő kettős-hibrid kísérletek segítségével sikerül fizikai kapcsolatokat kimutatnunk a Rev1-Rev7, Rad5-Rad18, Rad5-Ubc13, és a Rad18-PCNA fehérjék között. Az interakciók jelentőségének a megértéséhez a fenti fehérjekomplexeket tisztítottuk majd enzimatikusan jellemeztük, és megvizsgáltuk, hogy melyik fehérje lehet a Rad18 ubiquitin ligáz szubsztrátja. A Rad6-Rad18 enzim-komplex segítségével sikerült a PCNA replikációs fehérjét monoubiquitinálnunk, melynek tisztítása után jellemeztük aktiváló hatását a hibaátíró DNS polimerázok szintetikus aktivitására. Az elért új eredmények alapján elindítottunk egy új kutatási vonalat is, melynek célja az élesztő Rad5 emberi homológjának az azonosítása és jellemzése és a human ubiquitinált PCNA szerepének megismerése a károsított DNS replikációjában. | Genomic DNA is subjected to damage by external environmental agents and endogenous metabolic byproducts. To rescue the replication fork stalled due to encountering unrepaired DNA lesions the RAD6-RAD18-dependent damage avoidance mechanisms have evolved, which can lead to either error-free or error-prone replication of damaged DNA. The goal of our project was to shed more light on the function of Saccharomyces cerevisiae RAD6, RAD18, RAD5, PCNA, UBC13, MMS2, REV7, and REV1 genes in the replication of damaged DNA. With tandem affinity purification of protein complexes, and yeast two-hybrid method, we have detected physical interaction between Rev1-Rev7, Rad5-Rad18, Rad5-Ubc13, and Rad18-PCNA proteins. To gain insight into the significance of the above interactions, we characterized the enzymatic activities of the above complexes, and examined whether these proteins can be subject for Rad18-dependent ubiquitylation. We have managed to monoubiquitylate PCNA by Rad6-Rad18 enzyme in vitro and after purifying Ub-PCNA, we characterized its stimulatory effect on translesion synthesis polymerases. Based on these results, we have initiated a new study, as well, aiming to characterize the human homologue of yeast Rad5 protein and to unravel the function of PCNA ubiquitylation in damage bypass in human cells

Manganese is a strong specific activator of the rna synthetic activity of human polη

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

Selective Metal Ion Utilization Contributes to the Transformation of the Activity of Yeast Polymerase eta from DNA Polymerization toward RNA Polymerization

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

The Zn-Finger of Saccharomyces cerevisiae Rad18 and Its Adjacent Region Mediate Interaction with Rad5

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

Role of Double-Stranded DNA Translocase Activity of Human HLTF in Replication of Damaged DNA▿ §

Unrepaired DNA lesions can block the progression of the replication fork, leading to genomic instability and cancer in higher-order eukaryotes. In Saccharomyces cerevisiae, replication through DNA lesions can be mediated by translesion synthesis DNA polymerases, leading to error-free or error-prone damage bypass, or by Rad5-mediated template switching to the sister chromatid that is inherently error free. While translesion synthesis pathways are highly conserved from yeast to humans, very little is known of a Rad5-like pathway in human cells. Here we show that a human homologue of Rad5, HLTF, can facilitate fork regression and has a role in replication of damaged DNA. We found that HLTF is able to reverse model replication forks, a process which depends on its double-stranded DNA translocase activity. Furthermore, from analysis of isolated dually labeled chromosomal fibers, we demonstrate that in vivo, HLTF promotes the restart of replication forks blocked at DNA lesions. These findings suggest that HLTF can promote error-free replication of damaged DNA and support a role for HLTF in preventing mutagenesis and carcinogenesis, providing thereby for its potential tumor suppressor role