Faculty Bibliography

How DNA lesions in nucleosomes are recognized for global genome nucleotide excision repair (GG-NER) remains poorly understood, and the roles that histone tails may play remains to be established. Histone H3 and H4 N-terminal tails are of particular interest as their acetylation states are important in regulating nucleosomal functions in transcription, replication and repair. In particular the H3 tail has been the focus of recent attention as a site for the interaction with XPC, the GG-NER lesion recognition factor. Here we have investigated how the structure and dynamics of the DNA lesion cis-B[a]P-dG, derived from the environmental carcinogen benzo[a]pyrene (B[a]P), is impacted by the presence of flanking H3 and H4 tails. This lesion is well-repaired by GG-NER, and adopts a base-displaced/intercalated conformation in which the lesion partner C is displaced into the major groove. We used molecular dynamics simulations to obtain structural and dynamic characterizations for this lesion positioned in nucleosomal DNA so that it is bracketed by the H3 and H4 tails. The H4 tail was studied in unacetylated and acetylated states, while the H3 tail was unacetylated, its state when it binds XPC (Kakumu, Nakanishi et al., 2017). Our results reveal that upon acetylation, the H4 tail is released from the DNA surface; the H3 tail then forms a pocket that induces flipping and capture of the displaced lesion partner base C. This reveals synergistic effects of the behavior of the two tails. We hypothesize that the dual capability of the H3 tail to sense the displaced lesion partner base and to bind XPC could foster recognition of this lesion by XPC for initiation of GG-NER in nucleosomes.

BACKGROUND:, showing that its chronic expression in a p53-deficient environment causes genomic instability by deregulation of the replication licensing machinery. RESULTS:can further fuel genomic instability by suppressing the repair capacity of low- and high-fidelity pathways that deal with nucleotide abnormalities. Consequently, fewer single nucleotide substitutions (SNSs) occur, while formation of highly deleterious DNA double-strand breaks (DSBs) is enhanced, crafting a characteristic mutational signature landscape. Guided by the mutational signatures formed, we find that the DSBs are repaired by Rad52-dependent break-induced replication (BIR) and single-strand annealing (SSA) repair pathways. Conversely, the error-free synthesis-dependent strand annealing (SDSA) repair route is deficient. Surprisingly, Rad52 is activated transcriptionally in an E2F1-dependent manner, rather than post-translationally as is common for DNA repair factor activation. CONCLUSIONS:expression rewires the repair process and identifies Rad52 as a source of genomic instability and a candidate therapeutic target.

Background/UNASSIGNED:adenine-DNA glycosylase (MutY/MicA) protects cells against oxidative stress-induced mutagenesis by removing adenine which is mispaired with 7,8-dihydro-8-oxoguanine (8oxoG) in the base excision repair pathway. However, MutY does not discriminate between template and newly synthesized DNA strands. Therefore the ability to remove A from 8oxoG•A mispair, which is generated via misincorporation of an 8-oxo-2'-deoxyguanosine-5'-triphosphate precursor during DNA replication and in which A is the template base, can induce A•T→C•G transversions. Furthermore, it has been demonstrated that human MUTYH, homologous to the bacterial MutY, might be involved in the aberrant processing of ultraviolet (UV) induced DNA damage. Methods/UNASSIGNED:strains were measured. Results/UNASSIGNED:strains. Discussion/UNASSIGNED:These findings indicate that the bacterial MutY is not involved in the aberrant DNA repair of UV-induced DNA damage.

Benzo[a]pyrene (BP) is a carcinogen in cigarette smoke which, after metabolic activation, can react with the exocyclic N 2 amino group of guanine to generate four stereoisomeric BP-N 2-dG adducts. Rev1 is unique among translesion synthesis DNA polymerases in employing a protein-template-directed mechanism of DNA synthesis opposite undamaged and damaged guanine. Here we report high-resolution structures of yeast Rev1 with three BP-N 2-dG adducts, namely the 10S (+)-trans-BP-N 2-dG, 10R (+)-cis-BP-N 2-dG, and 10S ( - )-cis-BP-N 2-dG. Surprisingly, in all three structures, the bulky and hydrophobic BP pyrenyl residue is entirely solvent-exposed in the major groove of the DNA. This is very different from the adduct alignments hitherto observed in free or protein-bound DNA. All complexes are well poised for dCTP insertion. Our structures provide a view of cis-BP-N 2-dG adducts in a DNA polymerase active site, and offer a basis for understanding error-free replication of the BP-derived stereoisomeric guanine adducts.Benzo[a]pyrene (BP) is a carcinogen in cigarette smoke that upon metabolic activation reacts with guanine. Here, the authors present the structures of the translesion DNA synthesis polymerase Rev1 in complex with three of the four possible stereoisomeric BP-N 2 -dG adducts, which gives insights how Rev1 achieves error-free replication.

The eukaryotic global genomic nucleotide excision repair (GG-NER) pathway is the major mechanism that removes most bulky and some nonbulky lesions from cellular DNA. There is growing evidence that certain DNA lesions are repaired slowly or are entirely resistant to repair in cells, tissues, and in cell extract model assay systems. It is well established that the eukaryotic DNA lesion-sensing proteins do not detect the damaged nucleotide, but recognize the distortions/destabilizations in the native DNA structure caused by the damaged nucleotides. In this article, the nature of the structural features of certain bulky DNA lesions that render them resistant to NER, or cause them to be repaired slowly, is compared to that of those that are good-to-excellent NER substrates. Understanding the structural features that distinguish NER-resistant DNA lesions from good NER substrates may be useful for interpreting the biological significance of biomarkers of exposure of human populations to genotoxic environmental chemicals. NER-resistant lesions can survive to replication and cause mutations that can initiate cancer and other diseases. Furthermore, NER diminishes the efficacy of certain chemotherapeutic drugs, and the design of more potent pharmaceuticals that resist repair can be advanced through a better understanding of the structural properties of DNA lesions that engender repair-resistance.

The most common, oxidatively generated lesion in cellular DNA is 8-oxo-7,8-dihydroguanine, which can be oxidized further to yield highly mutagenic spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) in DNA. In human cell-free extracts, both lesions can be excised by base excision repair and global genomic nucleotide excision repair. However, it is not known if these lesions can be removed by transcription-coupled DNA repair (TCR), a pathway that clears lesions from DNA that impede RNA synthesis. To determine if Sp or Gh impedes transcription, which could make each a viable substrate for TCR, either an Sp or a Gh lesion was positioned on the transcribed strand of DNA under the control of a promoter that supports transcription by human RNA polymerase II. These constructs were incubated in HeLa nuclear extracts that contained active RNA polymerase II, and the resulting transcripts were resolved by denaturing polyacrylamide gel electrophoresis. The structurally rigid Sp strongly blocks transcription elongation, permitting 1.6 +/- 0.5% nominal lesion bypass. In contrast, the conformationally flexible Gh poses less of a block to human RNAPII, allowing 9 +/- 2% bypass. Furthermore, fractional lesion bypass for Sp and Gh is minimally affected by glycosylase activity found in the HeLa nuclear extract. These data specifically suggest that both Sp and Gh may well be susceptible to TCR because each poses a significant block to human RNA polymerase II progression. A more general principle is also proposed: Conformational flexibility may be an important structural feature of DNA lesions that enhances their transcriptional bypass.

The xeroderma pigmentosum C protein complex (XPC) recognizes a variety of environmentally induced DNA lesions and is the key in initiating their repair by the nucleotide excision repair (NER) pathway. When bound to a lesion, XPC flips two nucleotide pairs that include the lesion out of the DNA duplex, yielding a productively bound complex that can lead to successful lesion excision. Interestingly, the efficiencies of NER vary greatly among different lesions, influencing their toxicity and mutagenicity in cells. Though differences in XPC binding may influence NER efficiency, it is not understood whether XPC utilizes different mechanisms to achieve productive binding with different lesions. Here, we investigated the well-repaired 10R-(+)-cis-anti-benzo[a]pyrene-N2-dG (cis-B[a]P-dG) DNA adduct in a duplex containing normal partner C opposite the lesion. This adduct is derived from the environmental pro-carcinogen benzo[a]pyrene and is likely to be encountered by NER in the cell. We have extensively investigated its binding to the yeast XPC orthologue, Rad4, using umbrella sampling with restrained molecular dynamics simulations and free energy calculations. The NMR solution structure of this lesion in duplex DNA has shown that the dC complementary to the adducted dG is flipped out of the DNA duplex in the absence of XPC. However, it is not known whether the "pre-flipped" base would play a role in its recognition by XPC. Our results show that Rad4 first captures the displaced dC, which is followed by a tightly coupled lesion-extruding pathway for productive binding. This binding path differs significantly from the one deduced for the small cis-syn cyclobutane pyrimidine dimer lesion opposite mismatched thymines [ Mu , H. , ( 2015 ) Biochemistry , 54 ( 34 ), 5263 - 7 ]. The possibility of multiple paths that lead to productive binding to XPC is consistent with the versatile lesion recognition by XPC that is required for successful NER.

It is generally believed that the mammalian nucleotide excision repair pathway removes DNA helix-distorting bulky DNA lesions, while small non-bulky lesions are repaired by base excision repair (BER). However, recent work demonstrates that the oxidativly generated guanine oxidation products, spiroimininodihydantoin (Sp), 5-guanidinohydantoin (Gh), and certain intrastrand cross-linked lesions, are good substrates of NER and BER pathways that compete with one another in human cell extracts. The oxidation of guanine by peroxynitrite is known to generate 5-guanidino-4-nitroimidazole (NIm) which is structurally similar to Gh, except that the 4-nitro group in NIm is replaced by a keto group in Gh. However, unlike Gh, NIm is an excellent substrate of BER, but not of NER. These and other related results are reviewed and discussed in this article.

Histone tails in nucleosomes play critical roles in regulation of many biological processes, including chromatin compaction, transcription, and DNA repair. Moreover, post-translational modifications, notably lysine acetylation, are crucial to these functions. While the tails have been intensively studied, how the structures and dynamics of tails are impacted by the presence of a nearby bulky DNA lesion is a frontier research area, and how these properties are impacted by tail lysine acetylation remains unexplored. To obtain molecular insight, we have utilized all atom 3 mus molecular dynamics simulations of nucleosome core particles (NCPs) to determine the impact of a nearby DNA lesion, 10S (+)-trans-anti-B[a]P-N2-dG-the major adduct derived from the procarcinogen benzo[a]pyrene-on H2B tail behavior in unacetylated and acetylated states. We similarly studied lesion-free NCPs to investigate the normal properties of the H2B tail in both states. In the lesion-free NCPs, charge neutralization upon lysine acetylation causes release of the tail from the DNA. When the lesion is present, it stably engulfs part of the nearby tail, impairing the interactions between DNA and tail. With the tail in an acetylated state, the lesion still interacts with part of it, although unstably. The lesion's partial entrapment of the tail should hinder the tail from interacting with other nucleosomes, and other proteins such as acetylases, deacetylases, and acetyl-lysine binding proteins, and thus disrupt critical tail-governed processes. Hence, the lesion would impede tail functions modulated by acetylation or deacetylation, causing aberrant chromatin structures and impaired biological transactions such as transcription and DNA repair.

Oxidatively generated guanine radicals in DNA can undergo various nucleophilic reactions including the formation of C8-guanine cross-links with adjacent or nearby N3-thymines in DNA in the presence of O2. These G[8-3]T lesions have been identified in the DNA of human cells exposed to oxidative stress, and are most likely genotoxic if not removed by cellular defence mechanisms. The abilities of several representative polymerases to bypass the G[8-3]T lesions in two different sequence contexts, G*T* and G*CT*, were assessed in vitro. The polymerase BF (bacillus fragment) from Bacillus stearothermophilus, the Y-family archaeal polymerases Dpo4 from Sulfolobus sulfataricus P2, and human DNA pol κ and pol η were selected for the study. The A-family polymerase BF was strongly blocked, while relatively weak translesion synthesis was observed in the case of Y-family polymerases Dpo4 and pol κ. Primer extension catalyzed by pol η was also partially stalled at various positions at or near the G[8-3]T cross-linked bases, but a significant and distributive primer extension was observed beyond the sites of the lesions with the efficiency being consistently greater in the case of G*CT* than in the case of G*T* lesions. The results obtained with pol η are compared with translesion synthesis past other intrastrand cross-linked lesions with previously published results of others that include the isomeric G[8-5m]T lesions generated by ionizing radiation, the cis-syn cyclobutane pyrimidine dimer and the 6-4 photoproduct generated by UV irradiation, and the Pt-G*G* lesions derived from the reactions of the chemotherapeutic agent cisplatin with DNA.