Papers by Justyna McIntyre
Journal of Molecular Biology, 2007
Saccharomyces cerevisiae Rad30 is the homolog of human DNA polymerase eta whose inactivation lead... more Saccharomyces cerevisiae Rad30 is the homolog of human DNA polymerase eta whose inactivation leads to the cancer-prone syndrome xeroderma pigmentosum variant. Both human and yeast polymerase eta are responsible for error-free bypass of UV-induced cis-syn pyrimidine dimers and several other DNA lesions. Here we show, using yeast strains expressing TAP-tagged Rad30, that the level of this protein is posttranslationally regulated via ubiquitination and proteasome-mediated degradation. The half-life of Rad30 is 20 min and it increases due to proteasomal defects. Mutations inactivating components of the Skp1/ cullin/ F-box (SCF) ubiquitin ligase complex: Skp1 and the F-box protein Ufo1 stabilize Rad30. Our results indicate also that ultraviolet irradiation causes transient stabilization of Rad30, which leads, in turn, to temporary accumulation of this polymerase in the cell. We conclude that proteolysis plays an important role in regulating the cellular abundance of Rad30. These results are the first indication of a role for controlled proteasomal degradation in modulating cellular level of translesion DNA polymerase in eukaryotes.
Polymerase iota - an odd sibling among Y family polymerases
DNA Repair, Feb 1, 2020
DNA polymerase iota (polι) belongs to the Y-family of DNA polymerases that are involved in DNA da... more DNA polymerase iota (polι) belongs to the Y-family of DNA polymerases that are involved in DNA damage tolerance through their role in translesion DNA synthesis. Like all other Y-family polymerases, polι interacts with proliferating cell nuclear antigen (PCNA), Rev1, ubiquitin and ubiquitinated-PCNA and is also ubiquitinated itself. Here, we report that Polι also interacts with the p300 acetyltransferase and is acetylated. The primary acetylation site is K550, located in the Rev1-interacting region. However, K550 amino acid substitutions have no effect on Polι's ability to interact with Rev1. Interestingly, we find that acetylation of Polι significantly and specifically increases in response to S N 2 alkylating agents and to a lower extent to s N 1 alkylating and oxidative agents. As we have not observed acetylation of Polι's closest paralogue, DNA polymerase eta (Polη), with which Polι shares many functional similarities, we believe that this modification might exclusively regulate yet to be determined, and separate function(s) of Polι. The transfer of an acetyl group from acetyl coenzyme A to a specific site on a protein is one of the major posttranslational modifications and one of many that modify lysine residues 1,2. Lysine acetylation was first discovered in histones and its significance in transcription control has been demonstrated 3,4. Besides transcription, acetylation regulates many other cellular processes including the cell cycle, proliferation, apoptosis, DNA recombination, stress response and DNA repair 5-7. Lysine acetylation can have a profound and diverse impact on modified proteins as it can influence protein stability, localization, enzymatic activity, as well as DNA and protein binding 8-11. Acetylation is a dynamic modification catalyzed by acetyltransferases that can be reversed by deacetylases. The lysine acetyltransferases (KATs) are grouped in three major families and one of them, p300/ CREB-binding protein (CBP), consists of just two members, p300 and CBP 12. Mammalian p300 and CBP are paralogs sharing 86% amino acid identity in their aminotransferase domain and while they are conserved in metazoans, they do not have detectable sequence homology with other KATs 12,13. The enzymes possess a bromodomain that recognizes acetylated substrates and multiple other non-catalytic domains involved in protein binding 14. Additionally, a non-canonical, but functional RING domain, connects the enzymes with ubiquitination processes 15,16. p300/CBP interact with over 400 proteins and act as network hubs in different cellular pathways, often in complexes controlling transcriptional activation 17. Defects in these acetyltransferases have been linked to human diseases, including several types of cancer, heart malfunction, diabetes mellitus, as well as Rubinstein-Taybi syndrome, which is characterized by developmental abnormalities and cancer predisposition 17,18. On the other hand, due to the fact that p300/CBP are involved in the regulation of many tumor-relevant proteins including p53, c-myc, or BRCA1, many therapeutic strategies targeting p300/CBP are under investigation [reviewed in 19-22 ]. Despite the high homology between p300 and CBP, there is accumulating evidence to suggest that CBP and p300 are not fully redundant but, due to differential association with other proteins, or diversity in their substrate specificity, also have unique roles in vivo [reviewed in 18 ]. p300/CBP-directed lysine acetylation seems to play an important and diverse role in DNA replication and the DNA damage response. The p300/CBP-acetylated proteins are engaged in DNA damage recognition, signaling and most DNA repair pathways [ 23,24 , reviewed in 5 ]. However, until recently, there is little indication for the involvement of p300/CBP in the regulation of DNA damage tolerance mechanisms. Our previous report was the first to suggest such a possibility 25. We proposed that p300 acetyltransferase inhibition influences polyubiquitination of DNA polymerase iota (Polι), a non-canonical polymerase involved in DNA translesion synthesis (TLS).
Current Genetics, Feb 19, 2020
Y-family DNA polymerases mediate DNA damage tolerance via translesion synthesis (TLS). Because of... more Y-family DNA polymerases mediate DNA damage tolerance via translesion synthesis (TLS). Because of the intrinsically error-prone nature of these enzymes, their activities are regulated at several levels. Here, we demonstrate the common regulation of the cellular abundance of Y-family polymerases, polymerase eta (Pol eta), and Rev1, in response to DNA damage at various stages of the cell cycle. UV radiation influenced polymerase abundance more when cells were exposed in S-phase than in G1-or G2-phases. We noticed two opposing effects of UV radiation in S-phase. On one hand, exposure to increasing doses of UV radiation at the beginning of this phase increasingly delayed S-phase progression. As a result, the accumulation of Pol eta and Rev1, which in nonirradiated yeast is initiated at the S/G2-phase boundary, was gradually shifted into the prolonged S-phase. On the other hand, the extent of polymerase accumulation was inversely proportional to the dose of irradiation, such that the accumulation was significantly lower after exposure to 80 J/m 2 in S-phase than after exposure to 50 J/m 2 or 10 J/m 2. The limitation of polymerase accumulation in S-phase-arrested cells in response to high UV dose was suppressed upon RAD9 (but not MRC1) deletion. Additionally, hydroxyurea, which activates mainly the Mrc1-dependent checkpoint, did not limit Pol eta or Rev1 accumulation in S-phase-arrested cells. The results show that the accumulation of Y-family TLS polymerases is limited in S-phase-arrested cells due to high levels of DNA damage and suggest a role of the Rad9 checkpoint protein in this process.
Posttranslational Regulation of Human DNA Polymerase ι
Journal of Biological Chemistry, 2015
Human DNA polymerases (pols) η and ι are Y- family DNA polymerase paralogs that facilitate transl... more Human DNA polymerases (pols) η and ι are Y- family DNA polymerase paralogs that facilitate translesion synthesis (TLS) past damaged DNA. Both polη and polι can be monoubiquitinated in vivo. Polη has been shown to be ubiquitinated at one primary site. When this site is unavailable, three nearby lysines, may become ubiquitinated. In contrast, mass spectrometry analysis of monoubiquitinated polι revealed that it is ubiquitinated at over 27 unique sites. Many of these sites are localized in different functional domains of the protein, including the catalytic polymerase domain, the PCNA-interacting region, the Rev1-interacting region, as well as its Ubiquitin Binding Motifs, UBM1 and UBM2. Polι monoubiquitination remains unchanged after cells are exposed to DNA damaging agents such as UV- light (generating UV-photoproducts), ethyl methanesulfonate (generating alkylation damage), mitomycin C (generating interstrand crosslinks), or potassium bromate (generating direct oxidative DNA damage). However, when exposed to naphthoquinones, such as menadione and plumbagin, which cause indirect oxidative damage through mitochondrial dysfunction, polι becomes transiently polyubiquitinated via K11- and K48- linked chains of ubiquitin and subsequently targeted for degradation. Polyubiquitination does not occur as a direct result of the perturbation of the redox cycle, as no polyubiquitination was observed after treatment with rotenone, or antimycin A, which inhibit mitochondrial electron transport. Interestingly, polyubiquitination was observed after the inhibition of the lysine acetyltransferase, KATB3/p300. We hypothesize that the formation of polyubiquitination chains attached to polι occurs via the interplay between lysine acetylation and ubiquitination of ubiquitin itself at K11- and K48- rather than oxidative damage per se.
Posttranslational Regulation of Human DNA Polymerase ι
The Journal of biological chemistry, Jan 14, 2015
Human DNA polymerases (pols) η and ι are Y- family DNA polymerase paralogs that facilitate transl... more Human DNA polymerases (pols) η and ι are Y- family DNA polymerase paralogs that facilitate translesion synthesis (TLS) past damaged DNA. Both polη and polι can be monoubiquitinated in vivo. Polη has been shown to be ubiquitinated at one primary site. When this site is unavailable, three nearby lysines, may become ubiquitinated. In contrast, mass spectrometry analysis of monoubiquitinated polι revealed that it is ubiquitinated at over 27 unique sites. Many of these sites are localized in different functional domains of the protein, including the catalytic polymerase domain, the PCNA-interacting region, the Rev1-interacting region, as well as its Ubiquitin Binding Motifs, UBM1 and UBM2. Polι monoubiquitination remains unchanged after cells are exposed to DNA damaging agents such as UV- light (generating UV-photoproducts), ethyl methanesulfonate (generating alkylation damage), mitomycin C (generating interstrand crosslinks), or potassium bromate (generating direct oxidative DNA damage)...
DNA repair, Jan 18, 2015
Posttranslational modification of proteins often controls various aspects of their cellular funct... more Posttranslational modification of proteins often controls various aspects of their cellular function. Indeed, over the past decade or so, it has been discovered that posttranslational modification of lysine residues plays a major role in regulating translesion DNA synthesis (TLS) and perhaps the most appreciated lysine modification is that of ubiquitination. Much of the recent interest in ubiquitination stems from the fact that proliferating cell nuclear antigen (PCNA) was previously shown to be specifically ubiquitinated at K164 and that such ubiquitination plays a key role in regulating TLS. In addition, TLS polymerases themselves are now known to be ubiquitinated. In the case of human polymerase η, ubiquitination at four lysine residues in its C-terminus appears to regulate its ability to interact with PCNA and modulate TLS. Within the past few years, advances in global proteomic research have revealed that many proteins involved in TLS are, in fact, subject to a previously under...
The steady-state level and stability of TLS polymerase eta are cell cycle dependent in the yeast S. cerevisiae
DNA Repair, 2015
Polymerase eta (Pol eta) is a ubiquitous translesion DNA polymerase that is capable of bypassing ... more Polymerase eta (Pol eta) is a ubiquitous translesion DNA polymerase that is capable of bypassing UV-induced pyrimidine dimers in an error-free manner. However, this specialized polymerase is error prone when synthesizing through an undamaged DNA template. In Saccharomyces cerevisiae, both depletion and overproduction of Pol eta result in mutator phenotypes. Therefore, regulation of the cellular abundance of this enzyme is of particular interest. However, based on the investigation of variously tagged forms of Pol eta, mutually contradictory conclusions have been reached regarding the stability of this polymerase in yeast. Here, we optimized a protocol for the detection of untagged yeast Pol eta and established that the half-life of the native enzyme is 80±14min in asynchronously growing cultures. Experiments with synchronized cells indicated that the cellular abundance of this translesion polymerase changes throughout the cell cycle. Accordingly, we show that the stability of Pol eta, but not its mRNA level, is cell cycle stage dependent. The half-life of the polymerase is more than fourfold shorter in G1-arrested cells than in those at G2/M. Our results, in concert with previous data for Rev1, indicate that cell cycle regulation is a general property of Y family TLS polymerases in S. cerevisiae.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2006
Besides its role as a major recycler of unfolded or otherwise damaged intracellular proteins, the... more Besides its role as a major recycler of unfolded or otherwise damaged intracellular proteins, the 26S proteasome functions as a regulator of many vital cellular processes and is postulated as a target for antitumor drugs. It has previously been shown that dysfunction of the catalytic core of the 26S proteasome, the 20S proteasome, causes a moderate increase in the frequency of spontaneous mutations in yeast [A. Podlaska, J. McIntyre, A. Skoneczna, E. Sledziewska-Gojska, The link between proteasome activity and postreplication DNA repair in Saccharomyces cerevisiae. Mol. Microbiol. 49 (2003) 1321-1332. Here we show the results of genetic analysis, which indicate that the mutator phenotype caused by the deletion of UMP1, encoding maturase of 20S proteasome, involves members of the RAD6 epistasis group. The great majority of mutations occurring spontaneously in yeast cells deficient in 20S proteasome function are connected with the unique Rad6/Rad18-dependent error-prone translesion DNA synthesis (TLS) requiring the activities of both TLS polymerases: Pol and Pol . Our results suggest the involvement of proteasomal activity in the limitation of this unique error-prone TLS mechanism in wild-type cells. On the other hand, we found that the mutator phenotypes caused by deficiency in Rad18 and Rad6, are largely alleviated by defects in proteasome activities. Since the mutator phenotypes produced by deletion of RAD6 and RAD18 require Pol and Siz1/Ubc9-dependent sumoylation of PCNA, our results suggest that proteasomal dysfunction limits sumoylation-dependent error-prone activity of Pol . Taken together, our findings strongly support the idea that proteolytic activity is involved in modulating the balance between TLS mechanisms functioning during DNA replication in S. cerevisiae.
Nucleic Acids Research, 2013
Human DNA polymerases g and i are best characterized for their ability to facilitate translesion ... more Human DNA polymerases g and i are best characterized for their ability to facilitate translesion DNA synthesis (TLS). Both polymerases (pols) co-localize in 'replication factories' in vivo after cells are exposed to ultraviolet light and this co-localization is mediated through a physical interaction between the two TLS pols. We have mapped the polg-i interacting region to their respective ubiquitin-binding domains (UBZ in polg and UBM1 and UBM2 in poli), and demonstrate that ubiquitination of either TLS polymerase is a prerequisite for their physical and functional interaction. Importantly, while monoubiquitination of polg precludes its ability to interact with proliferating cell nuclear antigen (PCNA), it enhances its interaction with poli. Furthermore, a poli-ubiquitin chimera interacts avidly with both polg and PCNA. Thus, the ubiquitination status of polg, or poli plays a key regulatory function in controlling the protein partners with which each polymerase interacts, and in doing so, determines the efficiency of targeting the respective polymerase to stalled replication forks where they facilitate TLS.
Molecular Microbiology, 2011
Mms2, in concert with Ubc13 and Rad5, is responsible for polyubiquitination of replication proces... more Mms2, in concert with Ubc13 and Rad5, is responsible for polyubiquitination of replication processivity factor PCNA. This modification activates recombination-like DNA damage-avoidance mechanisms, which function in an error-free manner. Cells deprived of Mms2, Ubc13 or Rad5 exhibit mutator phenotypes as a result of the channelling of premutational DNA lesions to often error-prone translesion DNA synthesis (TLS). Here we show that Siz1mediated PCNA SUMOylation is required for the stimulation of this TLS, despite the presence of PCNA monoubiquitination. The stimulation of spontaneous mutagenesis by Siz1 in cells carrying rad5 and/or mms2 mutations is connected with the known role of PCNA SUMOylation in the inhibition of Rad52-mediated recombination. However, following UV irradiation, Siz1 is engaged in additional, as yet undefined, mechanisms controlling genetic stability at the replication fork. We also demonstrate that in the absence of PCNA SUMOylation, Mms2-Ubc13 and Rad5 may, independently of each other, function in the stimulation of TLS. Based on this finding and on an analysis of the epistatic relationships between SIZ1, MMS2 and RAD5, with respect to UV sensitivity, we conclude that PCNA SUMOylation is responsible for the functional differences between the Mms2 and Rad5 homologues of Saccharomyces cerevisiae and Schizosaccharomyces pombe.
Molecular Microbiology, 2003
We have shown previously that deletion of the Saccharomyces cerevisiae UMP1 gene encoding the 20S... more We have shown previously that deletion of the Saccharomyces cerevisiae UMP1 gene encoding the 20S proteasome maturase causes sensitivity to UV radiation. In the current report, we have extended this finding to show that mutations specifically compromising chymotrypsin-like or trypsin-like activity of 20S proteasome peptidases also result in increased UV sensitivity. We have also established that mutations affecting proteasome activity, namely ump1 D D D D , pre2-K108R and pup1-T20A , result in spontaneous and UVinduced mutator phenotypes. To elucidate the origin of these DNA repair phenotypes of the proteasomal mutants, we performed epistasis analysis, with respect to UV sensitivity, using yeast strains with the UMP1 deletion in different DNA repair backgrounds. We show that UMP1 is not epistatic to RAD23 and RAD2 , which are involved in the nucleotide excision repair (NER) pathway. Instead, our results indicate that UMP1 as well as PUP1 and PRE2 (encoding catalytic subunits of 20S proteasome) belong to an epistatic group of genes functioning in post-replication DNA repair (PRR) and are hypostatic to RAD18 , which, in complex with RAD6 , plays a central role in PRR. We also show that UMP1 is epistatic to REV3 and RAD30 , although the relationship of UMP1 with these genes is different.
Current Genetics, 2007
Ump1 is responsible for maturation of the catalytic core of the 26S proteasome. Dysfunction of Um... more Ump1 is responsible for maturation of the catalytic core of the 26S proteasome. Dysfunction of Ump1 causes an increase in the frequency of spontaneous mutations in Saccharomyces cerevisiae. In this study we analyze the spectrum of mutations occurring spontaneously in yeast deWcient in Ump1 by use of the SUP4-o system. Single base substitutions predominate among the mutations analyzed (73 of the 91 alterations examined). Two major classes are GC to TA transversions and GC to AT transitions (»50 and »30% of base substitutions, respectively). Besides base substitutions, almost all the major types of sequence alterations are represented. The speciWcity and distribution of mutations occurring in the ump1 strain are unique compared to the spectra previously established for other yeast mutators. However, the proWle of mutations arising in this strain is similar to that observed in wild type. The same similarity has previously been reported for yeast deWcient in Mms2, a protein involved in Rad6-dependent postreplication DNA repair (PRR). The speciWcity of the mutator eVect caused by ump1 is discussed in light of the proposed role of the proteasome activity in the regulation of the PRR mechanisms.
Journal of Molecular Biology, 2007
Saccharomyces cerevisiae Rad30 is the homolog of human DNA polymerase eta whose inactivation lead... more Saccharomyces cerevisiae Rad30 is the homolog of human DNA polymerase eta whose inactivation leads to the cancer-prone syndrome xeroderma pigmentosum variant. Both human and yeast polymerase eta are responsible for error-free bypass of UV-induced cis-syn pyrimidine dimers and several other DNA lesions. Here we show, using yeast strains expressing TAP-tagged Rad30, that the level of this protein is post-translationally regulated via ubiquitination and proteasome-mediated degradation. The half-life of Rad30 is 20 min and it increases due to proteasomal defects. Mutations inactivating components of the Skp1/cullin/ F-box (SCF) ubiquitin ligase complex: Skp1 and the F-box protein Ufo1 stabilize Rad30. Our results indicate also that ultraviolet irradiation causes transient stabilization of Rad30, which leads, in turn, to temporary accumulation of this polymerase in the cell. We conclude that proteolysis plays an important role in regulating the cellular abundance of Rad30. These results are the first indication of a role for controlled proteasomal degradation in modulating cellular level of translesion DNA polymerase in eukaryotes.
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Papers by Justyna McIntyre