A károsodott DNS replikációjának mechanizmusa és szabályozása: a mutagenezis és karcinogenezis háttere = Regulation and mechanism of replication of damaged DNA: implications for mutagenesis and carcinogenesis

A kijavítatlan DNS hibák gyakran gátolják a DNS replikációt. Saccharomyces cerevisiae-ben a károsított DNS replikációja történhet a hibák direkt átírására specializálódott DNS polimerázokkal illetve az ún. Rad5-függő posztreplikációs mechanizmus segítségével, amely az újonnan szintetizálódott szál a DNS hibával szemben kialakult diszkontinuitásait szünteti meg. Míg a polimerázok közreműködésével történő hibaátírás részletesen jellemzett ismereteink a posztreplikációs mechanizmusról igen korlátozottak. Projektünk célja az volt, hogy betekintést nyerjünk az élesztő Rad5-függő hibaátírásba. Kutatásaink során felfedeztük, hogy a Rad5 fehérjének egy speciális DNS helikáz aktivitása van, amely a replikációs villa visszafordítására specializálódott. Modell replikációs villa struktúrákon a Rad5 képes széttekerni mindkét minta/leány szálat majd hibridizálni egymáshoz a leány és a mintaszálakat. Ebből arra következtethetünk, hogy a Rad5 a DNS mintaszál-váltást katalizálva segíti elő a károsodott bázis hibamentes átírását. A továbbiakban sikerült azonosítanunk az élesztő Rad5 fehérjének két emberi homológját, a HLTF és SHPRH fehérjéket, is. A HLTF gyakran inaktiválódik vastagbél és gyomor rákban, míg az SHPRH a melanomák mintegy tíz százalékában mutálódik, amely tumor szuppresszor funkciójukra utalhat. Eredményeink szerint a HLTF és az SHPRH a károsított DNS hibamentes átírásában játszik kulcsszerepet, amely megmagyarázhatja tumor szuppresszor funkciójukat. | Lesions in the template DNA strand block the progression of the replication fork. In yeast, replication through DNA lesions is mediated by different Rad6-Rad18-dependent means, which include translesion synthesis and a Rad5-dependent postreplicational repair pathway that repairs the discontinuities that form in the DNA synthesized from damaged templates. Although translesion synthesis is well characterized, little is known about the Rad5-dependent pathway. The aim of our project has been to give insight into the yeast Rad5-dependent postreplicational repair pathway. We found that yeast Rad5 has a DNA helicase activity that is specialized for replication fork regression. On model replication fork structures, Rad5 concertedly unwinds and anneals the nascent and the parental strands without exposing extended single-stranded regions. These observations provide insight into the mechanism of postreplicational repair in which Rad5 action promotes template switching for error-free damage bypass. Also, we have identified and characterized two functional human homologues of yeast Rad5, the human HLTF and SHPRH proteins. HLTF is frequently inactivated in colorectal and gastric cancers while SHPRH is mutated in about ten percent of melanoma cell lines which has indicated a potential tumor suppressor function for these proteins. Our finding that HLTF and SHPRH play a role in error-free postreplication repair of damaged DNA is in keeping with its cancer-suppression role

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

Single Cell Analysis of Human RAD18-Dependent DNA Post-Replication Repair by Alkaline Bromodeoxyuridine Comet Assay

Damage to DNA can block replication progression resulting in gaps in the newly synthesized DNA. Cells utilize a number of post-replication repair (PRR) mechanisms such as the RAD18 controlled translesion synthesis or template switching to overcome the discontinuities formed opposite the DNA lesions and to complete DNA replication. Gaining more insights into the role of PRR genes promotes better understanding of DNA damage tolerance and of how their malfunction can lead to increased genome instability and cancer. However, a simple and efficient method to characterise gene specific PRR deficiencies at a single cell level has not been developed. Here we describe the so named BrdU comet PRR assay to test the contribution of human RAD18 to PRR at a single cell level, by which we kinetically characterized the consequences of the deletion of human RAD18 on the replication of UV-damaged DNA. Moreover, we demonstrate the capability of our method to evaluate PRR at a single cell level in unsynchronized cell population. © 2013 Mórocz et al

Role of PCNA-dependent stimulation of 3′-phosphodiesterase and 3′–5′ exonuclease activities of human Ape2 in repair of oxidative DNA damage

Human Ape2 protein has 3′ phosphodiesterase activity for processing 3′-damaged DNA termini, 3′–5′ exonuclease activity that supports removal of mismatched nucleotides from the 3′-end of DNA, and a somewhat weak AP-endonuclease activity. However, very little is known about the role of Ape2 in DNA repair processes. Here, we examine the effect of interaction of Ape2 with proliferating cell nuclear antigen (PCNA) on its enzymatic activities and on targeting Ape2 to oxidative DNA lesions. We show that PCNA strongly stimulates the 3′–5′ exonuclease and 3′ phosphodiesterase activities of Ape2, but has no effect on its AP-endonuclease activity. Moreover, we find that upon hydrogen-peroxide treatment Ape2 redistributes to nuclear foci where it colocalizes with PCNA. In concert with these results, we provide biochemical evidence that Ape2 can reduce the mutagenic consequences of attack by reactive oxygen species not only by repairing 3′-damaged termini but also by removing 3′-end adenine opposite from 8-oxoG. Based on these findings we suggest the involvement of Ape2 in repair of oxidative DNA damage and PCNA-dependent repair synthesis

Deletion of proteasomal subunit S5a/Rpn10/p54 causes lethality, multiple mitotic defects and overexpression of proteasomal genes in Drosophila melanogaster

The regulatory complex of the 26S proteasome is responsible for the selective recognition and binding of multiubiquitinated proteins. It was earlier shown that the subunit S5a/Rpn10/p54 of the regulatory complex is the only cellular protein capable of binding multiubiquitin chains in an in vitro overlay assay. The role of this subunit in substrate selection, however, is a subject of debate, following the observation that its deletion in Saccharomyces cerevisiae is not lethal and instead causes only a mild phenotype. To study the function of this subunit in higher eukaryotes, a mutant Drosophila strain was constructed by deleting the single copy gene encoding subunit S5a/Rpn10/p54. This deletion caused larval-pupal polyphasic lethality, multiple mitotic defects, the accumulation of higher multimers of ubiquitinated proteins and a huge accumulation of defective 26S proteasome particles. Deletion of the subunit S5a/Rpn10/p54 does not destabilise the regulatory complex and does not disturb the assembly of the regulatory complex and the catalytic core. The pupal lethality is a consequence of the depletion of the maternally provided 26S proteasome during the larval stages and a sudden increase in the proteasomal activity demands during the first few hours of pupal development. The huge accumulation of the fully assembled 26S proteasome in the deletion mutant and the lack of free subunits or partially assembled particles indicate that there is a highly coordinated accumulation of all the subunits of the 26S proteasome. This suggests that in higher eukaryotes, as with yeast, a feedback circuit coordinately regulates the expression of the proteasomal genes, and this adjusts the actual proteasome concentration in the cells according to the temporal and/or spatial proteolytic demand

Strand invasion by HLTF as a mechanism for template switch in fork rescue.

Stalling of replication forks at unrepaired DNA lesions can result in discontinuities opposite the damage in the newly synthesized DNA strand. Translesion synthesis or facilitating the copy from the newly synthesized strand of the sister duplex by template switching can overcome such discontinuities. During template switch, a new primer-template junction has to be formed and two mechanisms, including replication fork reversal and D-loop formation have been suggested. Genetic evidence indicates a major role for yeast Rad5 in template switch and that both Rad5 and its human orthologue, Helicase-like transcription factor (HLTF), a potential tumour suppressor can facilitate replication fork reversal. This study demonstrates the ability of HLTF and Rad5 to form a D-loop without requiring ATP binding and/or hydrolysis. We also show that this strand-pairing activity is independent of RAD51 in vitro and is not mechanistically related to that of another member of the SWI/SNF family, RAD54. In addition, the 3'-end of the invading strand in the D-loop can serve as a primer and is extended by DNA polymerase. Our data indicate that HLTF is involved in a RAD51-independent D-loop branch of template switch pathway that can promote repair of gaps formed during replication of damaged DNA

Def1 Promotes the Degradation of Pol3 for Polymerase Exchange to Occur During DNA-Damage–Induced Mutagenesis in Saccharomyces cerevisiae

The authors would like to thank Mark Hochstrasser for the MHY500 strain and Yasushi Saeki for the proteasome mutant and the corresponding wild-type strains. We also thank Sz. Minorits for technical assistance. This publication was also supported by the Dr. Rollin D. Hotchkiss Foundation. Funding: Wellcome Trust, 070247/Z/03/A. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD