Subpathway Of Nucleotide Excision Repair Research Articles

Interplay of the Tfb1 pleckstrin homology domain with Rad2 and Rad4 in transcription coupled and global genomic nucleotide excision repair.

Transcription-coupled repair (TCR) and global genomic repair (GGR) are two subpathways of nucleotide excision repair (NER). The TFIIH subunit Tfb1 contains a Pleckstrin homology domain (PHD), which was shown to interact with one PHD-binding segment (PB) of Rad4 and two PHD-binding segments (PB1 and PB2) of Rad2 in vitro. Whether and how the different Rad2 and Rad4 PBs interact with the same Tfb1 PHD, and whether and how they affect TCR and GGR within the cell remain mysterious. We found that Rad4 PB constitutively interacts with Tfb1 PHD, and the two proteins may function within one module for damage recognition in TCR and GGR. Rad2 PB1 protects Tfb1 from degradation and interacts with Tfb1 PHD at a basal level, presumably within transcription preinitiation complexes when NER is inactive. During a late step of NER, the interaction between Rad2 PB1 and Tfb1 PHD augments, enabling efficient TCR and GGR. Rather than interacting with Tfb1 PHD, Rad2 PB2 constrains the basal interaction between Rad2 PB1 and Tfb1 PHD, thereby weakening the protection of Tfb1 from degradation and enabling rapid augmentation of their interactions within TCR and GGR complexes. Our results shed new light on NER mechanisms.

Abstract 7369: Passenger mutations link cellular origins and transcriptional identity in human lung adenocarcinoma

Abstract Lineage plasticity hinders identification of cellular origin of cancers, as malignant cells cease to resemble benign cells from which they presumably evolved. Lung adenocarcinoma (LUAD), the most common primary lung cancer in United States is thought to arise from alveolar type II (AT2) cells. However recent mouse models have shown that LUADs may also arise from other cell types such as basal and club. Although such mouse models are considered to be gold standard for identifying cell of origin for cancers, they are generally limited to a specific genetic context and lack all cell types that are present in human lung. Thus, to better understand the cellular origins of LUAD, we utilized a publicly available lung scRNA-seq atlas from Human Cell Atlas, spanning over 584,000 cells from 107 individuals to investigate cell type specific passenger mutational footprints of transcriptional coupled repair process (a sub-pathway of nucleotide excision repair that preferentially corrects mutations in highly expressed genes) across 295 lung cancer WGS profiles. As most mutations in self-renewing tissues are thought to occur prior to the onset of tumorigenesis, genes that are most highly transcribed in the cell of origin would have fewer somatic single nucleotide variants (SNV). To test this hypothesis, our study correlated somatic mutational density with gene expression profiles of cell types to identify Alveolar Type 0 (AT0, newly identified cell type) cells to be most strongly associated with LUAD mutation density, suggesting that the ancestor of LUAD cells in tumor spent most of their non-cancerous time in AT0 state. Interestingly studies have shown that following DNA damage in AT2 cells, transition to AT1 occurs to repair the damage. However, if the damage is detrimental, the transition of AT2 to AT1 is stalled and the cells remain in AT0 (i.e., a transitionary) state. We propose that such stalling for a long period of time can give rise to LUAD. Further our study identified a subset of LUAD patients with non-AT2 (i.e., proximal) origin. In particular, we observed significantly higher KRAS mutations in distal origin LUAD patients compared to those in proximal origin LUADs. Finally, a subset of LUAD patients with distal cell of origin adopted a more proximal transcriptional identity. Extending our analysis to lung squamous cell carcinoma (LUSC) patients, we observed basal resting cells to be the Cell of Origin (COO) for LUSC. Our study provides a novel approach for identifying cell of origin for cancers and points to a complex interplay between origin and identify in lung adenocarcinoma evolution. Citation Format: Sukanya Panja, Padmaja Mantri, Juan Andrade Martinez, Marcin Imielinski. Passenger mutations link cellular origins and transcriptional identity in human lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7369.

Global repair is the primary nucleotide excision repair subpathway for the removal of pyrimidine-pyrimidone (6-4) damage from the Arabidopsis genome

Ultraviolet (UV) component of solar radiation impairs genome stability by inducing the formation of pyrimidine-pyrimidone (6-4) photoproducts [(6-4)PPs] in plant genomes. (6-4)PPs disrupt growth and development by interfering with transcription and DNA replication. To resist UV stress, plants employ both photoreactivation and nucleotide excision repair that excises oligonucleotide containing (6-4)PPs through two subpathways: global and transcription-coupled excision repair (TCR). Here, we analyzed the genome-wide excision repair-mediated repair of (6-4)PPs in Arabidopsis thaliana and found that (6-4)PPs can be repaired by TCR; however, the main subpathway to remove (6-4)PPs from the genome is global repair. Our analysis showed that open chromatin genome regions are more rapidly repaired than heterochromatin regions, and the repair level peaks at the promoter, transcription start site and transcription end site of genes. Our study revealed that the repair of (6-4)PP in plants showed a distinct genome-wide repair profile compared to the repair of other major UV-induced DNA lesion called cyclobutane pyrimidine dimers (CPDs).

The role of Transcription Factor IIH complex in nucleotide excision repair.

DNA damage occurs throughout life from a variety of sources, and it is imperative to repair damage in a timely manner to maintain genome stability. Thus, DNA repair mechanisms are a fundamental part of life. Nucleotide excision repair (NER) plays an important role in the removal of bulky DNA adducts, such as cyclobutane pyrimidine dimers from ultraviolet light or DNA crosslinking damage from platinum-based chemotherapeutics, such as cisplatin. A main component for the NER pathway is transcription factor IIH (TFIIH), a multifunctional, 10-subunit protein complex with crucial roles in both transcription and NER. In transcription, TFIIH is a component of the pre-initiation complex and is important for promoter opening and the phosphorylation of RNA Polymerase II (RNA Pol II). During repair, TFIIH is important for DNA unwinding, recruitment of downstream repair factors, and verification of the bulky lesion. Several different disease states can arise from mutations within subunits of the TFIIH complex. Most strikingly are xeroderma pigmentosum (XP), XP combined with Cockayne syndrome (CS), and trichothiodystrophy (TTD). Here, we summarize the recruitment and functions of TFIIH in the two NER subpathways, global genomic (GG-NER) and transcription-coupled NER (TC-NER). We will also discuss how TFIIH's roles in the two subpathways lead to different genetic disorders.

Rpb7 represses transcription-coupled nucleotide excision repair

Transcription-coupled repair (TCR) is a subpathway of nucleotide excision repair (NER) that is regulated by multiple facilitators, such as Rad26, and repressors, such as Rpb4 and Spt4/Spt5. How these factors interplay with each other and with core RNA polymerase II (RNAPII) remains largely unknown. In this study, we identified Rpb7, an essential RNAPII subunit, as another TCR repressor and characterized its repression of TCR in the AGP2, RPB2, and YEF3 genes, which are transcribed at low, moderate, and high rates, respectively. The Rpb7 region that interacts with the KOW3 domain of Spt5 represses TCR largely through the same common mechanism as Spt4/Spt5, as mutations in this region mildly enhance the derepression of TCR by spt4Δ only in the YEF3 gene but not in the AGP2 or RPB2 gene. The Rpb7 regions that interact with Rpb4 and/or the core RNAPII repress TCR largely independently of Spt4/Spt5, as mutations in these regions synergistically enhance the derepression of TCR by spt4Δ in all the genes analyzed. The Rpb7 regions that interact with Rpb4 and/or the core RNAPII may also play positive roles in other (non-NER) DNA damage repair and/or tolerance mechanisms, as mutations in these regions can cause UV sensitivity that cannot be attributed to derepression of TCR. Our study reveals a novel function of Rpb7 in TCR regulation and suggests that this RNAPII subunit may have broader roles in DNA damage response beyond its known function in transcription.

Distinctive Participation of Transcription-Coupled and Global Genome Nucleotide Excision Repair of Pyrimidine Dimers in the Transcribed Strand of Yeast rRNA Genes.

UV light causes the formation of pyrimidine dimers (PDs). Transcription-coupled (TC) nucleotide excision repair (NER) and global genome (GG) NER remove PDs from the transcribed strand (TS) of active genes and the inactive genome, respectively. TC-NER is triggered by elongating RNA polymerases that are blocked at PDs. The yeast rRNA genes are densely loaded with RNA polymerase-I. After UV irradiation, their density increases at the 5'-end of the gene, which results from continuous transcription initiation, followed by elongation and pausing/release at the first encountered PD, from the transcription start site. RNA polymerase-I posed at downstream PDs are released from the TS and are replaced by nucleosomes. Consequently, discrete chromatin structures are formed in the damaged transcribed rRNA genes. Singular assignation of the two NER sub-pathways could therefore be required to eliminate PDs from the TS. To advance our understanding of NER in the dynamic structure of transcribed chromatin, we investigated the repair of PDs at nucleotide resolution in separate rRNA gene coding regions. In the TS, the TC-NER efficiency reflected the density of RNA polymerase-I, and PDs were removed faster in the 5'-end than in the 3'-end of the gene. GG-NER removed PDs from the TS where RNA polymerase-I was transiently replaced by a nucleosome. The two NER sub-pathways inversely participated to remove PDs from the TS. In the non-TS of both nucleosome and non-nucleosome rRNA gene coding regions, GG-NER was solely responsible to remove UV-induced DNA lesions.

A disease-associated XPA allele interferes with TFIIH binding and primarily affects transcription-coupled nucleotide excision repair

XPA is a central scaffold protein that coordinates the assembly of repair complexes in the global genome (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER) subpathways. Inactivating mutations in XPA cause xeroderma pigmentosum (XP), which is characterized by extreme UV sensitivity and a highly elevated skin cancer risk. Here, we describe two Dutch siblings in their late forties carrying a homozygous H244R substitution in the C-terminus of XPA. They present with mild cutaneous manifestations of XP without skin cancer but suffer from marked neurological features, including cerebellar ataxia. We show that the mutant XPA protein has a severely weakened interaction with the transcription factor IIH (TFIIH) complex leading to an impaired association of the mutant XPA and the downstream endonuclease ERCC1-XPF with NER complexes. Despite these defects, the patient-derived fibroblasts and reconstituted knockout cells carrying the XPA-H244R substitution show intermediate UV sensitivity and considerable levels of residual GG-NER (~50%), in line with the intrinsic properties and activities of the purified protein. By contrast, XPA-H244R cells are exquisitely sensitive to transcription-blocking DNA damage, show no detectable recovery of transcription after UV irradiation, and display a severe deficiency in TC-NER-associated unscheduled DNA synthesis. Our characterization of a new case of XPA deficiency that interferes with TFIIH binding and primarily affects the transcription-coupled subpathway of nucleotide excision repair, provides an explanation of the dominant neurological features in these patients, and reveals a specific role for the C-terminus of XPA in TC-NER.

The Mfd protein is the transcription-repair coupling factor (TRCF) in Mycobacterium smegmatis

Invitro and invivo experiments with Escherichia coli have shown that the Mfd translocase is responsible for transcription-coupled repair, a subpathway of nucleotide excision repair involving the faster rate of repair of the transcribed strand than the nontranscribed strand. Even though the mfd gene is conserved in all bacterial lineages, there is only limited information on whether it performs the same function in other bacterial species. Here, by genome scale analysis of repair of UV-induced cyclobutane pyrimidine dimers, we find that the Mfd protein is the transcription-repair coupling factor in Mycobacterium smegmatis. This finding, combined with the inverted strandedness of UV-induced mutations in WT and mfd-E.coli and Bacillus subtilis indicate that the Mfd protein is the universal transcription-repair coupling factor in bacteria.

Nucleotide excision repair: a versatile and smart toolkit.

Nucleotide excision repair (NER) is a major pathway to deal with bulky adducts induced by various environmental toxins in all cellular organisms. The two sub-pathways of NER, global genome repair (GGR) and transcription-coupled repair (TCR), differ in the damage recognition modes. In this review, we describe the molecular mechanism of NER in mammalian cells, especially the details of damage recognition steps in both sub-pathways. We also introduce new sequencing methods for genome-wide mapping of NER, as well as recent advances about NER in chromatin by these methods. Finally, the roles of NER factors in repairing oxidative damages and resolving R-loops are discussed.

Crucial role and mechanism of transcription-coupled DNA repair in bacteria.

Transcription-coupled DNA repair (TCR) is presumed to be a minor sub-pathway of nucleotide excision repair (NER) in bacteria. Global genomic repair is thought to perform the bulk of repair independently of transcription. TCR is also believed to be mediated exclusively by Mfd-a DNA translocase of a marginal NER phenotype1-3. Here we combined in cellulo cross-linking mass spectrometry with structural, biochemical and genetic approaches to map the interactions within the TCR complex (TCRC) and to determine the actual sequence of events that leads to NER in vivo. We show that RNA polymerase (RNAP) serves as the primary sensor of DNA damage and acts as a platform for the recruitment of NER enzymes. UvrA and UvrD associate with RNAP continuously, forming a surveillance pre-TCRC. In response to DNA damage, pre-TCRC recruits a second UvrD monomer to form a helicase-competent UvrD dimer that promotes backtracking of the TCRC. The weakening of UvrD-RNAP interactions renders cells sensitive to genotoxic stress. TCRC then recruits a second UvrA molecule and UvrB to initiate the repair process. Contrary to the conventional view, we show that TCR accounts for the vast majority of chromosomal repair events; that is, TCR thoroughly dominates over global genomic repair. We also show that TCR is largely independent of Mfd. We propose that Mfd has an indirect role in this process: it participates in removing obstructive RNAPs in front of TCRCs and also in recovering TCRCs from backtracking after repair has been completed.

METTL14 facilitates global genome repair and suppresses skin tumorigenesis

Global genome repair (GGR), a subpathway of nucleotide excision repair, corrects bulky helix-distorting DNA lesions across the whole genome and is essential for preventing mutagenesis and skin cancer. Here, we show that METTL14 (methyltransferase-like 14), a critical component of the N6-methyladenosine (m6A) RNA methyltransferase complex, promotes GGR through regulating m6A mRNA methylation-mediated DDB2 translation and suppresses ultraviolet B (UVB) radiation-induced skin tumorigenesis. UVB irradiation down-regulates METTL14 protein through NBR1-dependent selective autophagy. METTL14 knockdown decreases GGR and DDB2 abundance. Conversely, overexpression of wild-type METTL14 but not its enzymatically inactive mutant increases GGR and DDB2 abundance. METTL14 knockdown decreases m6A methylation and translation of the DDB2 transcripts. Adding DDB2 reverses the GGR repair defect in METTL14 knockdown cells, indicating that METTL14 facilitates GGR through regulating DDB2 m6A methylation and translation. Similarly, knockdown of YTHDF1, an m6A reader promoting translation of m6A-modified transcripts, decreases DDB2 protein levels. Both METTL14 and YTHDF1 bind to the DDB2 transcript. In mice, skin-specific heterozygous METTL14 deletion increases UVB-induced skin tumorigenesis. Furthermore, METTL14 as well as DDB2 is down-regulated in human and mouse skin tumors and by chronic UVB irradiation in mouse skin, and METTL14 level is associated with the DDB2 level, suggesting a tumor-suppressive role of METTL14 in UVB-associated skin tumorigenesis in association with DDB2 regulation. Taken together, these findings demonstrate that METTL14 is a target for selective autophagy and acts as a critical epitranscriptomic mechanism to regulate GGR and suppress UVB-induced skin tumorigenesis.

Transcription coupled base excision repair in mammalian cells: So little is known and so much to uncover

Oxidized bases in the genome has been implicated in various human pathologies, including cancer, aging and neurological diseases. Their repair is initiated with excision by DNA glycosylases (DGs) in the base excision repair (BER) pathway. Among the five oxidized base-specific human DGs, OGG1 and NTH1 preferentially excise oxidized purines and pyrimidines, respectively, while NEILs remove both oxidized purines and pyrimidines. However, little is known about why cells possess multiple DGs with overlapping substrate specificities. Studies of the past decades revealed that some DGs are involved in repair of oxidized DNA base lesions in the actively transcribed regions. Preferential removal of lesions from the transcribed strands of active genes, called transcription-coupled repair (TCR), was discovered as a distinct sub-pathway of nucleotide excision repair; however, such repair of oxidized DNA bases had not been established until our recent demonstration of NEIL2’s role in TC-BER of the nuclear genome. We have shown that NEIL2 forms a distinct transcriptionally active, repair proficient complex. More importantly, we for the first time reconstituted TC-BER using purified components. These studies are important for characterizing critical requirement for the process. However, because NEIL2 cannot remove all types of oxidized bases, it is unlikely to be the only DNA glycosylase involved in TC-BER. Hence, we postulate TC-BER process to be universally involved in maintaining the functional integrity of active genes, especially in post-mitotic, non-growing cells. We further postulate that abnormal bases (e.g., uracil), and alkylated and other small DNA base adducts are also repaired via TC-BER. In this review, we have provided an overview of the various aspects of TC-BER in mammalian cells with the hope of generating significant interest of many researchers in the field. Further studies aimed at better understanding the mechanistic aspects of TC-BER could help elucidate the linkage of TC-BER deficiency to various human pathologies.

Distinct roles for RSC and SWI/SNF chromatin remodelers in genomic excision repair.

Nucleosomes are a significant barrier to the repair of UV damage because they impede damage recognition by nucleotide excision repair (NER). The RSC and SWI/SNF chromatin remodelers function in cells to promote DNA access by moving or evicting nucleosomes, and both have been linked to NER in yeast. Here, we report genome-wide repair maps of UV-induced cyclobutane pyrimidine dimers (CPDs) in yeast cells lacking RSC or SWI/SNF activity. Our data indicate that SWI/SNF is not generally required for NER but instead promotes repair of CPD lesions at specific yeast genes. In contrast, mutation or depletion of RSC subunits causes a general defect in NER across the yeast genome. Our data indicate that RSC is required for repair not only in nucleosomal DNA but also in neighboring linker DNA and nucleosome-free regions (NFRs). Although depletion of the RSC catalytic subunit also affects base excision repair (BER) of N-methylpurine (NMP) lesions, RSC activity is less important for BER in linker DNA and NFRs. Furthermore, our data indicate that RSC plays a direct role in transcription-coupled NER (TC-NER) of transcribed DNA. These findings help to define the specific genomic and chromatin contexts in which each chromatin remodeler functions in DNA repair, and indicate that RSC plays a unique function in facilitating repair by both NER subpathways.

Timely upstream events regulating nucleotide excision repair by ubiquitin-proteasome system: ubiquitin guides the way

The ubiquitin–proteasome system (UPS) plays crucial roles in regulation of multiple DNA repair pathways, including nucleotide excision repair (NER), which eliminates a broad variety of helix-distorting DNA lesions that can otherwise cause deleterious mutations and genomic instability. In mammalian NER, DNA damage sensors, DDB and XPC acting in global genomic NER (GG-NER), and, CSB and RNAPII acting in transcription-coupled NER (TC-NER) sub-pathways, undergo an array of post-translational ubiquitination at the DNA lesion sites. Accumulating evidence indicates that ubiquitination orchestrates the productive assembly of NER preincision complex by driving well-timed compositional changes in DNA damage-assembled sensor complexes. Conversely, the deubiquitination is also intimately involved in regulating the damage sensing aftermath, via removal of degradative ubiquitin modification on XPC and CSB to prevent their proteolysis for the factor recycling. This review summaries the relevant research efforts and latest findings in our understanding of ubiquitin-mediated regulation of NER and active participation by new regulators of NER, e.g., Cullin-Ring ubiquitin ligases (CRLs), ubiquitin-specific proteases (USPs) and ubiquitin-dependent segregase, valosin-containing protein (VCP)/p97. We project hypothetical step-by-step models in which VCP/p97-mediated timely extraction of damage sensors is integral to overall productive NER. The USPs and proteasome subtly counteract in fine-tuning the vital stability and function of NER damage sensors.

Structural basis for transcription complex disruption by the Mfd translocase.

Transcription-coupled repair (TCR) is a sub-pathway of nucleotide excision repair (NER) that preferentially removes lesions from the template-strand (t-strand) that stall RNA polymerase (RNAP) elongation complexes (ECs). Mfd mediates TCR in bacteria by removing the stalled RNAP concealing the lesion and recruiting Uvr(A)BC. We used cryo-electron microscopy to visualize Mfd engaging with a stalled EC and attempting to dislodge the RNAP. We visualized seven distinct Mfd-EC complexes in both ATP and ADP-bound states. The structures explain how Mfd is remodeled from its repressed conformation, how the UvrA-interacting surface of Mfd is hidden during most of the remodeling process to prevent premature engagement with the NER pathway, how Mfd alters the RNAP conformation to facilitate disassembly, and how Mfd forms a processive translocation complex after dislodging the RNAP. Our results reveal an elaborate mechanism for how Mfd kinetically discriminates paused from stalled ECs and disassembles stalled ECs to initiate TCR.

Regulation of UV damage repair in quiescent yeast cells

Non-growing quiescent cells face special challenges when repairing lesions produced by exogenous DNA damaging agents. These challenges include the global repression of transcription and translation and a compacted chromatin structure. We investigated how quiescent yeast cells regulated the repair of DNA lesions produced by UV irradiation. We found that UV lesions were excised and repaired in quiescent cells before their re-entry into S phase, and that lesion repair was correlated with high levels of Rad7, a recognition factor in the global genome repair sub-pathway of nucleotide excision repair (GGR-NER). UV exposure led to an increased frequency of mutations that included C->T transitions and T > A transversions. Mutagenesis was dependent on the error-prone translesion synthesis (TLS) DNA polymerase, Pol zeta, which was the only DNA polymerase present in detectable levels in quiescent cells. Across the genome of quiescent cells, UV-induced mutations showed an association with exons that contained H3K36 or H3K79 trimethylation but not with those bound by RNA polymerase II. Together, the data suggest that the distinct physiological state and chromatin structure of quiescent cells contribute to its regulation of UV damage repair.

Comparative study of cytotoxic effects induced by environmental genotoxins using XPC- and CSB-deficient human lymphoblastoid TK6 cells

BackgroundThe human genome is constantly exposed to numerous environmental genotoxicants. To prevent the detrimental consequences induced by the expansion of damaged cells, cellular protective systems such as nucleotide excision repair (NER) exist and serve as a primary pathway for repairing the various helix-distorting DNA adducts induced by genotoxic agents. NER is further divided into two sub-pathways, namely, global genomic NER (GG-NER) and transcription-coupled NER (TC-NER). Both NER sub-pathways are reportedly involved in the damage response elicited by exposure to genotoxins. However, how disruption of these sub-pathways impacts the toxicity of different types of environmental mutagens in human cells is not well understood.ResultsTo evaluate the role of NER sub-pathways on the cytotoxic effects of mutagens, we disrupted XPC and CSB to selectively inactivate GG-NER and TC-NER, respectively, in human lymphoblastoid TK6 cells, a standard cell line used in genotoxicity studies. Using these cells, we then comparatively assessed their respective sensitivities to representative genotoxic agents, including ultraviolet C (UVC) light, benzo [a] pyrene (B(a)P), 2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP), γ-ray, and 2-acetylaminofluorene (2-AAF). CSB−/− cells exhibited a hyper-sensitivity to UVC, B(a)P, and MeIQx. On the other hand, XPC−/− cells were highly sensitive to UVC, but not to B(a)P and MeIQx, compared with wild-type cells. In contrast with other genotoxins, the sensitivity of XPC−/− cells against PhIP was significantly higher than CSB−/− cells. The toxicity of γ-ray and 2-AAF was not enhanced by disruption of either XPC or CSB in the cells.ConclusionsBased on our findings, genetically modified TK6 cells appear to be a useful tool for elucidating the detailed roles of the various repair factors that exist to combat genotoxic agents, and should contribute to the improved risk assessment of environmental chemical contaminants.

Histone H4 H75E mutation attenuates global genomic and Rad26-independent transcription-coupled nucleotide excision repair.

Nucleotide excision repair (NER) consists of global genomic NER (GG-NER) and transcription coupled NER (TC-NER) subpathways. In eukaryotic cells, genomic DNA is wrapped around histone octamers (an H3–H4 tetramer and two H2A–H2B dimers) to form nucleosomes, which are well known to profoundly inhibit the access of NER proteins. Through unbiased screening of histone H4 residues in the nucleosomal LRS (loss of ribosomal DNA-silencing) domain, we identified 24 mutations that enhance or decrease UV sensitivity of Saccharomyces cerevisiae cells. The histone H4 H75E mutation, which is largely embedded in the nucleosome and interacts with histone H2B, significantly attenuates GG-NER and Rad26-independent TC-NER but does not affect TC-NER in the presence of Rad26. All the other histone H4 mutations, except for T73F and T73Y that mildly attenuate GG-NER, do not substantially affect GG-NER or TC-NER. The attenuation of GG-NER and Rad26-independent TC-NER by the H4H75E mutation is not due to decreased chromatin accessibility, impaired methylation of histone H3 K79 that is at the center of the LRS domain, or lowered expression of NER proteins. Instead, the attenuation is at least in part due to impaired recruitment of Rad4, the key lesion recognition and verification protein, to chromatin following induction of DNA lesions.

Roles of High Mobility Group Box Proteins in Nucleotide Excision Repair‐associated Processing of DNA Interstrand Crosslinks

Many traditional anti‐cancer chemotherapeutic agents induce DNA interstrand crosslinks (ICLs), lesions that form covalent bonds between the DNA strands. ICLs are highly cytotoxic if not properly repaired because they can block basic cellular processes such as transcription and replication. Nucleotide excision repair (NER) is a critical repair mechanism in processing ICLs in human cells. In addition to NER, ICLs are also processed through DNA double‐strand break repair pathways (e.g. homologous recombination) in S‐phase. We have previously demonstrated that the high mobility group box protein (HMGB1), a non‐histone architectural protein, can bind to site‐directed ICLs with high affinity and modulate error‐free repair processing of such lesions. In addition, we have found that HMGB1 associates with the NER damage recognition protein XPA in human cells and facilitates the recruitment of XPA to the damaged site. Further, we have demonstrated that HMGB1 can architecturally modify the damaged substrate by inducing negative supercoils, which may facilitate DNA damage processing by NER. We hypothesize that HMGB1 can modulate NER processing of ICLs by aiding NER damage‐recognition factors in locating damaged DNA. To test this hypothesis we are using a mutation‐reporter system to measure mutation frequencies and spectra generated by site‐directed ICLs in human cells. There are two NER subpathways that recognize DNA damage via different mechanisms. I am investigating in which pathway(s) HMGB1 is involved by utilizing various cell lines that are proficient or deficient for an essential recognition protein in each pathway. Additionally, we are investigating if HMGB2 and HMGB3, protein family members that are highly similar in structure to HMGB1, are also co‐factors in NER. Preliminary evidence suggests that HMGB1 may be involved in transcription coupled‐NER of site‐directed ICLs. If future evidence supports these data, this would indicate that HMGB1 could associate with transcription machinery and aid in recruiting other NER co‐factors, such as XPA. Future genetic, molecular biological, and biochemical assays will provide additional insight as to the roles of the HMGB proteins in ICL repair. The processing and repair of ICLs in human cells is not completely understood, and therefore a detailed study to define the molecular mechanisms of ICL processing is warranted. Identifying new proteins involved in ICL processing may lead to new pharmacological targets, which may help us to improve the outcome of cancer treatment.Support or Funding InformationNIH/NCI to K.V. (CA093729) and NSF GRFP to J.G.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evidence that Moderate Eviction of Spt5 and Promotion of Error-Free Transcriptional Bypass by Rad26 Facilitates Transcription Coupled Nucleotide Excision Repair

Transcription coupled repair (TC-NER) is a subpathway of nucleotide excision repair triggered by stalling of RNA polymerase at DNA lesions. It has been suspected that transcriptional misincorporations of certain nucleotides opposite lesions that result in irreversible transcription stalling might be important for TC-NER. However, the spectra of nucleotide misincorporations opposite UV photoproducts and how they are implicated in transcriptional stalling and TC-NER in the cell remain unknown. Rad26, a low abundant yeast protein, and its human homolog CSB have been proposed to facilitate TC-NER in part by positioning and stabilizing stalling of RNA polymerase II (RNAPII) at DNA lesions. Here, we found that substantial AMPs but no other nucleotides are transcriptionally misincoporated and extended opposite UV photoproducts and adjacent bases in Saccharomyces cerevisiae. Rad26 does not significantly affect either the misincorporation or extension of AMPs. At normally low or moderately increased levels, Rad26 promotes error-free transcriptional bypass and TC-NER of UV photoproducts. However, Rad26 completely loses these functions when it is overexpressed to ~1/3 the level of RNAPII molecules. Also, Rad26 does not directly displace RNAPII but constitutively evicts Spt5, a key transcription elongation factor and TC-NER repressor, from the chromatin. Our results indicate that transcriptional nucleotide misincorporation is not implicated in TC-NER, and moderate eviction of Spt5 and promotion of error-free transcriptional bypass of DNA lesions by Rad26 facilitates TC-NER.