Replicative polymerases (Pols) arrest at damaged DNA nucleotides, which induces ubiquitination of the DNA sliding clamp PCNA (PCNA-Ub) and DNA damage signaling. PCNA-Ub is associated with the... Show moreReplicative polymerases (Pols) arrest at damaged DNA nucleotides, which induces ubiquitination of the DNA sliding clamp PCNA (PCNA-Ub) and DNA damage signaling. PCNA-Ub is associated with the recruitment or activation of translesion synthesis (TLS) DNA polymerases of the Y family that can bypass the lesions, thereby rescuing replication and preventing replication fork collapse and consequent formation of double-strand DNA breaks. Here, we have used gene-targeted mouse embryonic fibroblasts to perform a comprehensive study of the in vivo roles of PCNA-Ub and of the Y family TLS Pols η, ι, κ, Rev1 and the B family TLS Polζ in TLS and in the suppression of DNA damage signaling and genome instability after exposure to UV light. Our data indicate that TLS Pols ι and κ and the N-terminal BRCT domain of Rev1, that previously was implicated in the regulation of TLS, play minor roles in TLS of DNA photoproducts. PCNA-Ub is critical for an early TLS pathway that replicates both strongly helix-distorting (6-4) pyrimidine-pyrimidone ((6-4)PP) and mildly distorting cyclobutane pyrimidine dimer (CPD) photoproducts. The role of Polη is mainly restricted to early TLS of CPD photoproducts, whereas Rev1 and, in particular, Polζ are essential for the bypass of (6-4)PP photoproducts, both early and late after exposure. Thus, structurally distinct photoproducts at the mammalian genome are bypassed by different TLS Pols in temporally different, PCNA-Ub-dependent and independent fashions. Show less
Temviriyanukul, P.; Hees-Stuivenberg, S. van; Delbos, F.; Jacobs, H.; Wind, N. de; Jansen, J.G. 2012
DNA lesions, induced by genotoxic compounds block the processive replication fork but can be bypassed by specialized translesion synthesis (TLS) DNA Polymerases (Pols). TLS safeguards the... Show moreDNA lesions, induced by genotoxic compounds block the processive replication fork but can be bypassed by specialized translesion synthesis (TLS) DNA Polymerases (Pols). TLS safeguards the completion of replication, albeit at the expense of nucleotide substitution mutations. We studied the in vivo role of individual TLS Pols in cellular responses to benzo[a]pyrene diolepoxide (BPDE), a polycyclic aromatic hydrocarbon, and 4-hydroxynonenal (4-HNE), a product of lipid peroxidation. To this aim, we used mouse embryonic fibroblasts with targeted disruptions in the TLS-associated Pols η, ι, κ and Rev1, as well as in Rev3, the catalytic subunit of TLS Polζ. After exposure, cellular survival, replication fork progression, DNA damage responses (DDR), and the induction of micronuclei was investigated. The results demonstrate that Rev1, Rev3 and, to a lesser extent, Polη are involved in TLS, DDR and the prevention of DNA breaks, in response to both agents. Conversely, Polκ and the N-terminal BRCT domain of Rev1 are specifically involved in TLS of BPDE-induced DNA damage. We furthermore describe a novel role of Polι in TLS of 4-HNE-induced DNA damage in vivo. We hypothesize that different sets of TLS polymerases act on structurally different genotoxic DNA lesions in vivo, thereby suppressing genomic instability associated with cancer. Our experimental approach may provide a significant contribution in delineating the molecular bases of the genotoxicity in vivo of different classes of DNA damaging agents. Show less
Diamant, N.; Hendel, A.; Vered, I.; Carell, T.; Reissner, T.; Wind, N. de; ... ; Livneh, Z. 2012
Genomes encode all RNAs required for life. For this simple reason the genome's stability is a prerequisite for maintaining the fitness of the cell, the organism and its progeny. Paradoxically, any... Show moreGenomes encode all RNAs required for life. For this simple reason the genome's stability is a prerequisite for maintaining the fitness of the cell, the organism and its progeny. Paradoxically, any enzymatic transaction at the DNA, including transcription itself, entails the risk of local destabilization of the DNA helix, thereby threatening genomic integrity. Particularly where transcription and replication meet the genome may be at an increased risk of nucleotide substitution mutations, deletions or rearrangements. This Extra-view sketches our understanding of the different threats that transcription imposes on genome stability. We will focus on recent work highlighting the role of DNA damage in transcription-associated mutagenesis (TAM) in mammalian cells. Furthermore we discuss the possible implications of TAM for human fitness and disease with an emphasis on carcinogenesis. In addition, we propose an updated nomenclature for the mechanistically different forms of TAM. Show less
Drost, M.; Zonneveld, J.B.M.; Dijk, L. van; Morreau, H.; Tops, C.M.; Vasen, H.F.A.; ... ; Wind, N. de 2010
The hereditary colon and endometrium cancer predisposition Lynch Syndrome (also called HNPCC) is caused by a germ-line mutation in one of the DNA mismatch repair (MMR) genes. A significant fraction... Show moreThe hereditary colon and endometrium cancer predisposition Lynch Syndrome (also called HNPCC) is caused by a germ-line mutation in one of the DNA mismatch repair (MMR) genes. A significant fraction of the gene alterations detected in suspected Lynch Syndrome patients is comprised of amino acid substitutions. The relevance for cancer risk of these variants is difficult to assess, as currently no time- and cost-effective, validated, and widely applicable functional assays for the measurement of MMR activity are available. Here we describe a rapid, cell-free, and easily quantifiable MMR activity assay for the diagnostic assessment of variants of the MLH1 MMR protein. This assay allows the parallel generation and functional analysis of a series of variants of the MLH1 protein in vitro using readily available, or preprepared, reagents. Using this assay we have tested 26 MLH1 variants and of these, 15 had lost activity. These results are in concordance with those obtained from first-generation assays and with in silico and pathology data. After its multifocal technical and clinical validation this assay could have great impact for the diagnosis and counseling of carriers of an MLH1 variant and their relatives. Show less
Hendriks, G.; Calleja, F.; Besaratinia, A.; Vrieling, H.; Pfeifer, G.P.; Mullenders, L.H.F.; ... ; Wind, N. de 2010
Skin cancer is the most ubiquitous cancer type in the Caucasian population, and its incidence is increasing rapidly [1]. Transcribed proliferation-related genes in dermal stem cells are targets for... Show moreSkin cancer is the most ubiquitous cancer type in the Caucasian population, and its incidence is increasing rapidly [1]. Transcribed proliferation-related genes in dermal stem cells are targets for the induction of ultraviolet light (UV)-induced mutations that drive carcinogenesis. We have recently found that transcription of a gene increases its mutability by UV in mammalian stem cells, suggesting a role of transcription in skin carcinogenesis [2]. Here we show that transcription-associated UV-induced nucleotide substitutions are caused by increased deamination of cytosines to uracil within photolesions at the transcribed strand, presumably at sites of stalled transcription complexes. Additionally, via an independent mechanism, transcription of UV-damaged DNA induces the generation of intragenic deletions. We demonstrate that transcription-coupled nucleotide excision repair (TC-NER) provides protection against both classes of transcription-associated mulagenesis. Combined, these results unveil the existence of two mutagenic pathways operating specifically at the transcribed DNA strand of active genes. Moreover, these results uncover a novel role for TC-NER in the suppression of UV-induced genome aberrations and provide a rationale for the efficient induction of apoptosis by stalled transcription complexes. Show less
Hendriks, G.; Calleja, F.; Besaratinia, A.; Vrieling, H.; Pfeifer, G.P.; Mullenders, L.H.F.; ... ; Wind, N. de 2010
Skin cancer is the most ubiquitous cancer type in the Caucasian population, and its incidence is increasing rapidly [1]. Transcribed proliferation-related genes in dermal stem cells are targets for... Show moreSkin cancer is the most ubiquitous cancer type in the Caucasian population, and its incidence is increasing rapidly [1]. Transcribed proliferation-related genes in dermal stem cells are targets for the induction of ultraviolet light (UV)-induced mutations that drive carcinogenesis. We have recently found that transcription of a gene increases its mutability by UV in mammalian stem cells, suggesting a role of transcription in skin carcinogenesis [2]. Here we show that transcription-associated UV-induced nucleotide substitutions are caused by increased deamination of cytosines to uracil within photolesions at the transcribed strand, presumably at sites of stalled transcription complexes. Additionally, via an independent mechanism, transcription of UV-damaged DNA induces the generation of intragenic deletions. We demonstrate that transcription-coupled nucleotide excision repair (TC-NER) provides protection against both classes of transcription-associated mulagenesis. Combined, these results unveil the existence of two mutagenic pathways operating specifically at the transcribed DNA strand of active genes. Moreover, these results uncover a novel role for TC-NER in the suppression of UV-induced genome aberrations and provide a rationale for the efficient induction of apoptosis by stalled transcription complexes. Show less
![The Vital Role of Polymerase zeta and REV1 in Mutagenic, but Not Correct, DNA Synthesis across Benzo[a]pyrene-dG and Recruitment of Polymerase zeta by REV1 to Replication-stalled Site](/sites/all/modules/custom/islandora_solr_search/images/defaultimg.png?solr_nav%5Bid%5D=17ae4bb093692fc6b9e8&solr_nav%5Bpage%5D=2&solr_nav%5Boffset%5D=6)
