Nucleolytic processing of abasic sites underlies PARP inhibitor hypersensitivity in ALC1-deficient BRCA mutant cancer cells.

Clinical success with poly (ADP-ribose) polymerase inhibitors (PARPi) is impeded by inevitable resistance and associated cytotoxicity. Depletion of Amplified in Liver Cancer 1 (ALC1), a chromatin-remodeling enzyme, can overcome these limitations by hypersensitizing BReast CAncer genes 1/2 (BRCA1/2) mutant cells to PARPi. Here, we demonstrate that PARPi hypersensitivity upon ALC1 loss is reliant on its role in promoting the repair of chromatin buried abasic sites. We show that ALC1 enhances the ability of the abasic site processing enzyme, Apurinic/Apyrimidinic endonuclease 1 (APE1) to cleave nucleosome-occluded abasic sites. However, unrepaired abasic sites in ALC1-deficient cells are readily accessed by APE1 at the nucleosome-free replication forks. APE1 cleavage leads to fork breakage and trapping of PARP1/2 upon PARPi treatment, resulting in hypersensitivity. Collectively, our studies reveal how cells overcome the chromatin barrier to repair abasic lesions and uncover cleavage of abasic sites as a mechanism to overcome limitations of PARPi.

Single-molecule analysis of purified proteins and nuclear extracts: Insights from 8-oxoguanine glycosylase 1.

By observing one molecule at a time, single-molecule studies can offer detailed insights about biomolecular processes including on rates, off rates, and diffusivity of molecules on strands of DNA. A recent technological advance (Single-molecule Analysis of DNA-binding proteins from Nuclear Extracts, SMADNE) has lowered the barrier to entry for single-molecule studies, and single-molecule dynamics can now be determined directly out of nuclear extracts, providing information in an intermediate environment between purified proteins in isolation and the heterogeneity of a nucleus. To compare and contrast the single-molecule DNA binding dynamics in nuclear extracts versus purified proteins, combined optical tweezers and fluorescence microscopy experiments were performed with purified GFP-tagged 8-oxoguanine glycosylase 1 (OGG1), purified GFP-OGG1 spiked into nuclear extracts, and nuclear extracts from human cells overexpressing GFP-OGG1. We observed differences in undamaged DNA binding during DNA damage search in each of the three conditions. Purified GFP-OGG1 engaged undamaged DNA for a weighted average lifetime of 5.7 s and 21% of these events underwent DNA diffusion after binding. However, unlike other glycosylases studied by SMADNE, OGG1 does not bind non-damaged DNA efficiently in nuclear extracts. In contrast, GFP-OGG1 binding dynamics on DNA substrates containing oxidative damage were relatively similar in all three conditions, with the weighted average binding lifetimes varying from 2.2 s in nuclear extracts to 7.8 s with purified GFP-OGG1 in isolation. Finally, we compared the purified protein and nuclear extract approaches for a catalytically dead OGG1 variant (GFP-OGG1-K249Q). This variant greatly increased the binding lifetime for oxidative DNA damage, with the weighted average lifetime for GFP-OGG1-249Q in nuclear extracts at 15.4 s vs 10.7 s for the purified protein. SMADNE will provide a new window of observation into the behavior of nucleic acid binding proteins only accessible by biophysicists trained in protein purification and protein labeling.

Single-molecule analysis of purified proteins and nuclear extracts: insights from 8-oxoguanine glycosylase 1.

By observing one molecule at a time, single-molecule studies can offer detailed insights about biomolecular processes including on rates, off rates, and diffusivity of molecules on strands of DNA. A recent technological advance (Single-molecule Analysis of DNA-binding proteins from Nuclear Extracts, SMADNE) has lowered the barrier to entry for single-molecule studies, and single-molecule dynamics can now be determined directly out of nuclear extracts, providing information in an intermediate environment between purified proteins in isolation and the heterogeneity of a nucleus. To compare and contrast the single-molecule DNA binding dynamics in nuclear extracts versus purified proteins, combined optical tweezers and fluorescence microscopy experiments were performed with purified GFP-tagged 8-oxoguanine glycosylase 1 (OGG1), purified GFP-OGG1 spiked into nuclear extracts, and nuclear extracts from human cells overexpressing GFP-OGG1. We observed differences in undamaged DNA binding during DNA damage search in each of the three conditions. Purified GFP-OGG1 engaged undamaged DNA for a weighted average lifetime of 5.7 s and 21% of these events underwent DNA diffusion after binding. However, unlike other glycosylases studied by SMADNE, OGG1 does not bind non-damaged DNA efficiently in nuclear extracts. In contrast, GFP-OGG1 binding dynamics on DNA substrates containing oxidative damage were relatively similar in all three conditions, with the weighted average binding lifetimes varying from 2.2 s in nuclear extracts to 7.8 s with purified GFP-OGG1 in isolation. Finally, we compared the purified protein and nuclear extract approaches for a catalytically dead OGG1 variant (GFP-OGG1-K249Q). This variant greatly increased the binding lifetime for oxidative DNA damage, with the weighted average lifetime for GFP-OGG1-249Q in nuclear extracts at 15.4 s vs 10.7 s for the purified protein. SMADNE will provide a new window of observation into the behavior of nucleic acid binding proteins only accessible by biophysicists trained in protein purification and protein labeling.

Single-molecule analysis reveals TDG exhibits multiple modes of linear diffusion to process 5-formylcytosine.

DNA methylation plays a key role in epigenetics, with 60-80% of CpG sites containing 5-methylcytosine. Base excision repair (BER) is suggested to be the main pathway involved in active DNA demethylation. 5-formylctyosine (5fC), an oxidized moiety of methylated cytosine, is recognized and removed by thymine DNA glycosylase (TDG) to generate an abasic site. TDG binds avidly to abasic sites and is product inhibited. Using single molecule fluorescence experiments, we saw TDG interact with DNA containing 5fC specifically and non-specifically with lifetimes of 72.9 and 7.5 seconds, respectively. These results indicate that TDG cleaves the 5fC and stays bound for an extended time at the generated abasic site. Mean squared displacement analysis and a two color TDG experiment indicate that TDG exhibits multiple modes of linear diffusion, including hopping and sliding, in search of a lesion. The catalytically crippled variants, N140A and R275A/L, have a reduced binding lifetime compared to wild type and Mean Squared Displacement (MSD) analysis indicates that R275L/A moves on the DNA with a faster diffusivity. These results indicate that mutating R275, but not N140 interferes with damage recognition by TDG. Our findings give insight into how TDG searches for its lesions in long stretches of undamaged DNA.

Generation of Recombinant Nucleosomes Containing Site-Specific DNA Damage.

Eukaryotic DNA exists in chromatin, where the genomic DNA is packaged into a fundamental repeating unit known as the nucleosome. In this chromatin environment, our genomic DNA is constantly under attack by exogenous and endogenous stressors that can lead to DNA damage. Importantly, this DNA damage must be repaired to prevent the accumulation of mutations and ensure normal cellular function. To date, most in-depth biochemical studies of DNA repair proteins have been performed in the context of free duplex DNA. However, chromatin can serve as a barrier that DNA repair enzymes must navigate in order find, access, and process DNA damage in the cell. To facilitate future studies of DNA repair in chromatin, we describe a protocol for generating nucleosome containing site-specific DNA damage that can be utilized for a variety of in vitro applications. This protocol describes several key steps including how to generate damaged DNA oligonucleotides, the expression and purification of recombinant histones, the refolding of histone complexes, and the reconstitution of nucleosomes containing site-specific DNA damage. These methods will enable researchers to generate nucleosomes containing site-specific DNA damage for extensive biochemical and structural studies of DNA repair in the nucleosome.

New insights into DNA polymerase mechanisms provided by time-lapse crystallography.

DNA polymerases play central roles in DNA replication and repair by catalyzing template-directed nucleotide incorporation. Recently time-lapse X-ray crystallography, which allows one to observe reaction intermediates, has revealed numerous and unexpected mechanistic features of DNA polymerases. In this article, we will examine recent new discoveries that have come from time-lapse crystallography that are currently transforming our understanding of the structural mechanisms used by DNA polymerases. Among these new discoveries are the binding of a third metal ion within the polymerase active site, the mechanisms of translocation along the DNA, the presence of new fidelity checkpoints, a novel pyrophosphatase activity within the active site, and the mechanisms of pyrophosphorolysis.

Structural basis for APE1 processing DNA damage in the nucleosome.

Genomic DNA is continually exposed to endogenous and exogenous factors that promote DNA damage. Eukaryotic genomic DNA is packaged into nucleosomes, which present a barrier to accessing and effectively repairing DNA damage. The mechanisms by which DNA repair proteins overcome this barrier to repair DNA damage in the nucleosome and protect genomic stability is unknown. Here, we determine how the base excision repair (BER) endonuclease AP-endonuclease 1 (APE1) recognizes and cleaves DNA damage in the nucleosome. Kinetic assays determine that APE1 cleaves solvent-exposed AP sites in the nucleosome with 3 - 6 orders of magnitude higher efficiency than occluded AP sites. A cryo-electron microscopy structure of APE1 bound to a nucleosome containing a solvent-exposed AP site reveal that APE1 uses a DNA sculpting mechanism for AP site recognition, where APE1 bends the nucleosomal DNA to access the AP site. Notably, additional biochemical and structural characterization of occluded AP sites identify contacts between the nucleosomal DNA and histone octamer that prevent efficient processing of the AP site by APE1. These findings provide a rationale for the position-dependent activity of BER proteins in the nucleosome and suggests the ability of BER proteins to sculpt nucleosomal DNA drives efficient BER in chromatin.

Mechanism of nucleotide discrimination by the translesion synthesis polymerase Rev1.

Rev1 is a translesion DNA synthesis (TLS) polymerase involved in the bypass of adducted-guanine bases and abasic sites during DNA replication. During damage bypass, Rev1 utilizes a protein-template mechanism of DNA synthesis, where the templating DNA base is evicted from the Rev1 active site and replaced by an arginine side chain that preferentially binds incoming dCTP. Here, we utilize X-ray crystallography and molecular dynamics simulations to obtain structural insight into the dCTP specificity of Rev1. We show the Rev1 R324 protein-template forms sub-optimal hydrogen bonds with incoming dTTP, dGTP, and dATP that prevents Rev1 from adopting a catalytically competent conformation. Additionally, we show the Rev1 R324 protein-template forms optimal hydrogen bonds with incoming rCTP. However, the incoming rCTP adopts an altered sugar pucker, which prevents the formation of a catalytically competent Rev1 active site. This work provides novel insight into the mechanisms for nucleotide discrimination by the TLS polymerase Rev1.

Reprogramming CBX8-PRC1 function with a positive allosteric modulator.

Canonical targeting of Polycomb repressive complex 1 (PRC1) to repress developmental genes is mediated by cell-type-specific, paralogous chromobox (CBX) proteins (CBX2, 4, 6, 7, and 8). Based on their central role in silencing and their dysregulation associated with human disease including cancer, CBX proteins are attractive targets for small-molecule chemical probe development. Here, we have used a quantitative and target-specific cellular assay to discover a potent positive allosteric modulator (PAM) of CBX8. The PAM activity of UNC7040 antagonizes H3K27me3 binding by CBX8 while increasing interactions with nucleic acids. We show that treatment with UNC7040 leads to efficient and selective eviction of CBX8-containing PRC1 from chromatin, loss of silencing, and reduced proliferation across different cancer cell lines. Our discovery and characterization of UNC7040 not only reveals the most cellularly potent CBX8-specific chemical probe to date, but also corroborates a mechanism of Polycomb regulation by non-specific CBX nucleotide binding activity.

Visualizing Rev1 catalyze protein-template DNA synthesis.

During DNA replication, replicative DNA polymerases may encounter DNA lesions, which can stall replication forks. One way to prevent replication fork stalling is through the recruitment of specialized translesion synthesis (TLS) polymerases that have evolved to incorporate nucleotides opposite DNA lesions. Rev1 is a specialized TLS polymerase that bypasses abasic sites, as well as minor-groove and exocyclic guanine adducts. Lesion bypass is accomplished using a unique protein-template mechanism in which the templating base is evicted from the DNA helix and the incoming dCTP hydrogen bonds with an arginine side chain of Rev1. To understand the protein-template mechanism at an atomic level, we employed a combination of time-lapse X-ray crystallography, molecular dynamics simulations, and DNA enzymology on the Saccharomyces cerevisiae Rev1 protein. We find that Rev1 evicts the templating base from the DNA helix prior to binding the incoming nucleotide. Binding the incoming nucleotide changes the conformation of the DNA substrate to orient it for nucleotidyl transfer, although this is not coupled to large structural changes in Rev1 like those observed with other DNA polymerases. Moreover, we found that following nucleotide incorporation, Rev1 converts the pyrophosphate product to two monophosphates, which drives the reaction in the forward direction and prevents pyrophosphorolysis. Following nucleotide incorporation, the hydrogen bonds between the incorporated nucleotide and the arginine side chain are broken, but the templating base remains extrahelical. These postcatalytic changes prevent potentially mutagenic processive synthesis by Rev1 and facilitate dissociation of the DNA product from the enzyme.

The EZH2 SANT1 domain is a histone reader providing sensitivity to the modification state of the H4 tail.

SANT domains are found in a number of chromatin regulators. They contain approximately 50 amino acids and have high similarity to the DNA binding domain of Myb related proteins. Though some SANT domains associate with DNA others have been found to bind unmodified histone tails. There are two SANT domains in Enhancer of Zeste 2 (EZH2), the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2), of unknown function. Here we show that the first SANT domain (SANT1) of EZH2 is a histone binding domain with specificity for the histone H4 N-terminal tail. Using NMR spectroscopy, mutagenesis, and molecular modeling we structurally characterize the SANT1 domain and determine the molecular mechanism of binding to the H4 tail. Though not important for histone binding, we find that the adjacent stimulation response motif (SRM) stabilizes SANT1 and transiently samples its active form in solution. Acetylation of H4K16 (H4K16ac) or acetylation or methylation of H4K20 (H4K20ac and H4K20me3) are seen to abrogate binding of SANT1 to H4, which is consistent with these modifications being anti-correlated with H3K27me3 in-vivo. Our results provide significant insight into this important regulatory region of EZH2 and the first characterization of the molecular mechanism of SANT domain histone binding.

Engagement of DNA and H3K27me3 by the CBX8 chromodomain drives chromatin association.

Polycomb repressive complex 1 (PRC1) is critical for mediating gene repression during development and adult stem cell maintenance. Five CBX proteins, CBX2,4,6,7,8, form mutually exclusive PRC1 complexes and are thought to play a role in the association of PRC1 with chromatin. Specifically, the N-terminal chromodomain (CD) in the CBX proteins is thought to mediate specific targeting to methylated histones. For CBX8, however, the chromodomain has demonstrated weak affinity and specificity for methylated histones in vitro, leaving doubt as to its role in CBX8 chromatin association. Here, we investigate the function of the CBX8 CD in vitro and in vivo. We find that the CD is in fact a major driver of CBX8 chromatin association and determine that this is driven by both histone and previously unrecognized DNA binding activity. We characterize the structural basis of histone and DNA binding and determine how they integrate on multiple levels. Notably, we find that the chromatin environment is critical in determining the ultimate function of the CD in CBX8 association.

Reading More than Histones: The Prevalence of Nucleic Acid Binding among Reader Domains.

The eukaryotic genome is packaged into the cell nucleus in the form of chromatin, a complex of genomic DNA and histone proteins. Chromatin structure regulation is critical for all DNA templated processes and involves, among many things, extensive post-translational modification of the histone proteins. These modifications can be "read out" by histone binding subdomains known as histone reader domains. A large number of reader domains have been identified and found to selectively recognize an array of histone post-translational modifications in order to target, retain, or regulate chromatin-modifying and remodeling complexes at their substrates. Interestingly, an increasing number of these histone reader domains are being identified as also harboring nucleic acid binding activity. In this review, we present a summary of the histone reader domains currently known to bind nucleic acids, with a focus on the molecular mechanisms of binding and the interplay between DNA and histone recognition. Additionally, we highlight the functional implications of nucleic acid binding in chromatin association and regulation. We propose that nucleic acid binding is as functionally important as histone binding, and that a significant portion of the as yet untested reader domains will emerge to have nucleic acid binding capabilities.

Covalent Modifications of Histone H3K9 Promote Binding of CHD3.

Chromatin remodeling is required for genome function and is facilitated by ATP-dependent complexes, such as nucleosome remodeling and deacetylase (NuRD). Among its core components is the chromodomain helicase DNA binding protein 3 (CHD3) whose functional significance is not well established. Here, we show that CHD3 co-localizes with the other NuRD subunits, including HDAC1, near the H3K9ac-enriched promoters of the NuRD target genes. The tandem PHD fingers of CHD3 bind histone H3 tails and posttranslational modifications that increase hydrophobicity of H3K9-methylation or acetylation (H3K9me3 or H3K9ac)-enhance this interaction. Binding of CHD3 PHDs promotes H3K9Cme3-nucleosome unwrapping in vitro and perturbs the pericentric heterochromatin structure in vivo. Methylation or acetylation of H3K9 uniquely alleviates the intra-nucleosomal interaction of histone H3 tails, increasing H3K9 accessibility. Collectively, our data suggest that the targeting of covalently modified H3K9 by CHD3 might be essential in diverse functions of NuRD.

Rapid Detection of Urinary Tract Infections via Bacterial Nuclease Activity.

Rapid and accurate bacterial detection methods are needed for clinical diagnostic, water, and food testing applications. The wide diversity of bacterial nucleases provides a rich source of enzymes that could be exploited as signal amplifying biomarkers to enable rapid, selective detection of bacterial species. With the exception of the use of micrococcal nuclease activity to detect Staphylococcus aureus, rapid methods that detect bacterial pathogens via their nuclease activities have not been developed. Here, we identify endonuclease I as a robust biomarker for E. coli and develop a rapid ultrasensitive assay that detects its activity. Comparison of nuclease activities of wild-type and nuclease-knockout E. coli clones revealed that endonuclease I is the predominant DNase in E. coli lysates. Endonuclease I is detectable by immunoblot and activity assays in uropathogenic E. coli strains. A rapid assay that detects endonuclease I activity in patient urine with an oligonucleotide probe exhibited substantially higher sensitivity for urinary tract infections than that reported for rapid urinalysis methods. The 3 hr turnaround time is much shorter than that of culture-based methods, thereby providing a means for expedited administration of appropriate antimicrobial therapy. We suggest this approach could address various unmet needs for rapid detection of E. coli.