Biology subjects

Freudenthal, B. D.

Publications and source records attributed to Freudenthal, B. D..

At least 19 recordsLinked to original sources

bioRxiv2026

XRCC1 Enables the Efficient Local Search for DNA Damage by DNA Polymerase Beta

Oxidative DNA damage is a common threat to genomic integrity, arising from endogenous metabolic processes and environmental exposures. If unrepaired, such oxidative DNA damage promotes mutagenesis and genomic instability. Cells counter this through base excision repair (BER), a multi-step pathway requiring the coordinated action of several proteins. Central to BER, DNA polymerase beta (pol ) locates single-nucleotide (1-nt) gaps and inserts the correct nucleotide, while x-ray repair cross-complementing 1 (XRCC1) is a scaffold protein that forms a stable complex with pol to coordinate BER factors at DNA damage. XRCC1 enhances BER efficiency, though the mechanism by which this occurs is unclear. Pol {beta} is proposed to be recruited to DNA damage by undamaged DNA scanning interactions, but this behavior has not yet been directly observed. Additionally, the influence of other BER proteins on pol recruitment, particularly XRCC1, remains unclear. Here, we used correlative optical tweezers-fluorescence microscopy to visualize DNA search and damage recognition by pol and XRCC1. We characterize each factor individually, examine their behavior as the pol -XRCC1 complex, and assess their interplay with apurinic/apyrimidinic endonuclease 1 (APE1), the enzyme upstream of pol in BER. We find that pol locates damage through 3D-diffusion, whereas XRCC1 exhibits both 3D- and 1D-diffusion. In combination, XRCC1 dramatically shifts pol {beta} search towards 1D-diffusion, enabling interrogation of non-damaged DNA using both search mechanisms. When both APE1 and pol are present, the pol -1nt gap complex is highly stable, with APE1 largely unable to disrupt the damage-bound pol . Together, these findings demonstrate that XRCC1 reshapes pol {beta} search behavior to promote efficient local damage recognition, providing a mechanistic basis for how BER factors coordinate lesion detection and processing to maintain genomic stability.

biochemistry↗

bioRxiv2026

A Dynamic Search Mechanism Enables APE1 to Identify AP-Sites in DNA

Apurinic/apyrimidinic (AP) sites are among the most frequent DNA lesions, arising thousands of times per cell each day. These AP-sites threaten genomic stability and, if left unrepaired, can lead to mutagenesis and human disease. The essential base excision repair enzyme Apurinic/Apyrimidinic Endonuclease I (APE1) initiates repair by cleaving DNA at AP-sites, yet how APE1 efficiently locates and recognizes these lesions within vast excesses of undamaged DNA has remained poorly understood. Using single-molecule imaging, we show that APE1 employs a dynamic search strategy that integrates 1D and 3D diffusion to rapidly scan DNA. On non-damaged DNA, APE1 undergoes fast diffusion, enabling efficient interrogation of large genomic regions within a single binding event. Upon encountering an AP-site, APE1 transitions from a mobile search state into a stationary, lesion-bound complex that is retained at the site of damage. Additional experiments with APE1 variants demonstrate that the intrinsically disordered N-terminal domain of APE1 supports 1D diffusion, whereas residue R177 stabilizes APE1 at the AP-site once recognized, and the catalytic residues D210 and E96 facilitate enzyme release after cleavage. Together, these findings define the molecular basis by which APE1 balances rapid genome surveillance with stable lesion engagement and timely release. More broadly, this work provides a mechanistic framework for how DNA repair enzymes efficiently locate and process rare lesions embedded within an excess of undamaged DNA. Significance StatementDNA repair enzymes face the formidable challenge of locating rare lesions hidden within vast stretches of undamaged DNA. The essential enzyme APE1 is tasked with identifying and initiating repair of AP-sites, a frequent DNA lesion, to maintain genome integrity. We show that APE1 overcomes this challenge by using a dynamic search mechanism that integrates rapid scanning with precise lesion recognition. Its unstructured N-terminal domain facilitates fast movement along non-damaged DNA, while specific active site residues stabilize engagement at AP-sites and release after catalysis. This coordination of diffusion, recognition, and turnover allows APE1 to efficiently survey the genome and repair AP-sites to maintain genome stability.

biochemistry↗

bioRxiv2025

Structure, dynamics, and processing of 8oxoG:A in the nucleosome

Eukaryotic genomic DNA is packaged into chromatin through a repeating unit known as the nucleosome. In this chromatin environment, DNA is constantly exposed to several sources of DNA damage, such as reactive oxygen species (ROS), which can lead to the formation of 8-oxo-7,8-dihydroguanine (8oxoG). 8oxoG can base pair with cytosine (8oxoG:C) or form a mutagenic base pair with adenine (8oxoG:A), which can lead to single base transversions if left unrepaired. To date, the structure and dynamics of these two possible 8oxoG base pairs in the nucleosome remain unclear. Furthermore, whether MutY homologue (MUTYH) excises 8oxoG:A base pairs in the nucleosome remains elusive. Here using a combination of cryogenic-electron microscopy, molecular dynamics simulations, and biochemistry we determined the structure and dynamics of 8oxoG:C and 8oxoG:A base pairs in the nucleosome and characterize MUTYH activity in nucleosomal DNA. We found that nucleosomal 8oxoG:C forms a stable base pair using its anti conformation, while nucleosomal 8oxoG:A forms a more dynamic base pair using its syn conformation that is unable to be processed by MUTYH. This work provides fundamental insight into the accommodation of oxidative damage in the nucleosome and how this damage contributes to increased mutagenic transversions in nucleosomal compared to linker DNA.

biochemistry↗

bioRxiv2025

Nucleosome unwrapping and PARP1 allostery drive affinities for chromatin and DNA breaks

Poly[ADP-ribose] polymerase 1 (PARP1) detects DNA strand breaks that occur in duplex DNA and chromatin. We employed correlative optical tweezers and fluorescence microscopy to quantify how single molecules of PARP1 identify single-strand breaks (i.e., nicks), undamaged nucleosome core particles (NCP) and NCPs containing DNA nicks. Fluorescently-tagged PARP1 or PARP2 from nuclear extracts bound nicks with nanomolar affinity but did not engage undamaged dsDNA regions. In contrast, PARP1 avidly bound undamaged NCPs, and partial NCP unwrapping induced by DNA tension significantly increased the on rate and affinity. Catalytically dead PARP1 or EB-47 inhibition greatly increased PARP1 affinity to DNA nicks and undamaged NCP, implicating a mechanism where PARP1 reverse allostery regulates PARP1 retention to undamaged chromatin. We also monitored ADP-ribosylation in real time upon PARP1 binding undamaged or nicked NCPs. These results provide key mechanistic insights into domain allostery and how pharmacological intervention alters PARP1 binding dynamics for therapeutic impacts.

biophysics↗

bioRxiv2025

APE1 active site residue Asn174 stabilizes the AP-site and is essential for catalysis

Apurinic/Apyrimidinic (AP)-sites are common and highly mutagenic DNA lesions that can arise spontaneously or as intermediates during Base Excision Repair (BER). The enzyme apurinic/apyrimidinic endonuclease 1 (APE1) initiates repair of AP-sites by cleaving the DNA backbone at the AP-site via its endonuclease activity. Here, we investigated the functional role of the APE1 active site residue N174 that contacts the AP-site during catalysis. We analyzed the effects of three rationally designed APE1 mutations that alter the hydrogen bonding potential, size, and charge of N174: N174A, N174D, and N174Q. We found impaired catalysis of the APE1N174A and APE1N174D mutants due to disruption of hydrogen bonding and electrostatic interactions between residue 174 and the AP-site. In comparison, the APE1N174Q mutant was less impaired due to retaining similar hydrogen bonding and electrostatic characteristics as N174 in wild-type APE1. Structures and computational simulations further revealed that the AP-site was destabilized within the active sites of the APE1N174A and APE1N174D mutants due to loss of hydrogen bonding between residue 174 and the AP-site. Cumulatively, we show that N174 stabilizes the AP-site within the APE1 active site through hydrogen bonding and electrostatic interactions to enable effective catalysis. These findings highlight the importance of N174 in APE1s function and provide new insights into the molecular mechanism by which APE1 processes AP-sites during DNA repair.

biochemistry↗

bioRxiv2024

Structure of Blm10:13S proteasome intermediate reveals parallel assembly pathways for the proteasome core particle

Proteasomes are formed by chaperone-assisted assembly of core particles (CPs) and regulatory particles (RPs). The CP chaperone dimer Pba1/Pba2 binds early to proteasome subunits, and is thought to be replaced by Blm10 to form Blm10:CP, which promotes ATP-independent degradation of disordered proteins. Here, we present evidence of distinct parallel assembly pathways for CP by solving five cryo-EM structures including a Blm10:13S pre-assembly intermediate. Our data conflict with the current model of Blm10 and Pba1/Pba2 sequential activity in a single assembly pathway, as we find their CP binding is mutually exclusive and both are present on early and late assembly intermediates. CP affinity for Pba1/Pba2 is reduced during maturation, promoting Pba1/Pba2 release. We find Blm10 undergoes no such affinity switch, suggesting this pathway predominantly yields mature Blm10-bound CP. Altogether, our findings conflict with the current paradigm of sequential CP binding to instead indicate parallel assembly pathways by Pba1/Pba2 and Blm10.

biochemistry↗

bioRxiv2024

Structural basis of gap-filling DNA synthesis in the nucleosome by DNA Polymerase β

Single-strand breaks (SSBs) are one of the most prevalent forms of DNA damage found in the chromatinized genome and are repaired by direct single-strand break repair (SSBR) or base excision repair (BER). DNA polymerase beta (Pol {beta}) is the primary enzyme responsible for processing the 1-nt gap intermediate in chromatin during SSBR and BER. However, the mechanism used by Pol {beta} to process a 1-nt gap in the context of the nucleosome and chromatin remains poorly understood. Here, we use biochemical assays and cryogenic electron microscopy (cryo-EM) to determine the kinetic and structural basis of gap-filling DNA synthesis in the nucleosome by Pol {beta}. Kinetic analysis identified that gap-filling DNA synthesis in the nucleosome by Pol {beta} is position-dependent, where solvent exposed 1-nt gaps are processed more efficiently than histone-occluded 1-nt gaps. A series of cryo-EM structures of Pol {beta} bound to a solvent-exposed 1-nt gap in the nucleosome reveal a global DNA sculpting mechanism for 1-nt gap recognition, which is mediated by sequential engagement of the Pol {beta} lyase domain and polymerase domain. Finally, cryo-EM structures of Pol {beta} bound to 1-nt gaps at two additional positions in the nucleosomal DNA define the structural basis for position-dependent nucleotide insertion in the nucleosome. This work establishes the mechanism used by Pol {beta} for processing 1-nt gaps in the nucleosome during SSBR and BER, providing fundamental insight into DNA repair in chromatin.

biochemistry↗

bioRxiv2024

Hierarchical determinants of the oxidation-induced mutational landscape in human cells.

8-oxoguanine (8-oxoG) is a common oxidative DNA lesion, which causes G>T substitutions that compose COSMIC single base substitution signature 18 (SBS18) in human cancers. Determinants of local and regional differences in 8-oxoG-induced mutability are currently unknown. To uncover factors influencing the topology of 8-oxoG-induced mutations, we assessed spontaneous and KBrO3-induced 8-oxoG mutagenesis in human cell lines. KBrO3 exposure produced a SBS18-like substitution spectrum and a distinct never-before reported INDEL signature that we also observed in human cancers. KBrO3-induced 8-oxoG lesions occurred with similar sequence preference as KBrO3-induced substitutions, indicating that the reactivity of specific reactive oxygen species (ROS) dictates the trinucleotide motif specificity for 8-oxoG-induced mutagenesis. While 8-oxoG lesions occurred relatively uniformly across chromatin states and nucleosomes, 8-oxoG-induced mutations occurred more frequently in more compact regions of the genome, within nucleosomal DNA, and at inward facing guanines within strongly positioned nucleosomes. Cryo-EM structures of OGG1 bound to nucleosomes indicate that these effects originate from OGG1s ability to flip outward positioned 8-oxoG lesions into the catalytic pocket with only minor alterations to nucleosome structure, while inward facing lesions occluded by the histone octamer are unrecognized. Mutation spectra from cells with DNA repair deficiencies revealed a hierarchical DNA repair network limiting 8-oxoG mutagenesis in human cells, where OGG1- and MUTY-mediated BER is supplemented by replication-associated factors participating in tolerance of 8-oxoG or derived repair intermediates (i.e. Pol {eta} and HMCES). Surprisingly, analysis of transcriptional asymmetry of KBrO3-induced mutations demonstrated transcription-coupled repair of 8-oxoG in Pol {eta}-deficient cells. Thus, radical chemistry, chromatin structures, and DNA repair processes combine to dictate the oxidative mutational landscape in human genomes.

cancer biology↗

bioRxiv2024

Molecular basis for the activation of the Fatty Acid Kinase complex of Staphylococcus aureus

Gram-positive bacteria utilize a Fatty Acid Kinase (FAK) complex to harvest fatty acids from the environment. The complex, consisting of the fatty acid kinase, FakA, and an acyl carrier protein, FakB, is known to impact virulence and disease outcomes. However, FAKs structure and enzymatic mechanism remain poorly understood. Here, we used a combination of modeling, biochemical, and cell-based approaches to establish critical details of FAK activity. Solved structures of the apo and ligand-bound FakA kinase domain captured the protein state through ATP hydrolysis. Additionally, targeted mutagenesis of an understudied FakA Middle domain identified critical residues within a metal-binding pocket that contribute to FakA dimer stability and protein function. Regarding the complex, we demonstrated nanomolar affinity between FakA and FakB and generated computational models of the complexs quaternary structure. Together, these data provide critical insight into the structure and function of the FAK complex which is essential for understanding its mechanism.

microbiology↗

bioRxiv2024

Biochemical and Structural Characterization of Fapy·dG Replication by Human DNA Polymerase β

N6-(2-deoxy-,{beta}-D-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido-pyrimidine (Fapy*dG) is formed from a common intermediate and in comparable amounts to the well-studied mutagenic DNA lesion 8-oxo-7,8-dihydro-2-deoxyguanosine (8-OxodGuo). Fapy*dG preferentially gives rise to G [->] T transversions and G [->] A transitions. However, the molecular basis by which Fapy*dG is processed by DNA polymerases during this mutagenic process remains poorly understood. To address this we investigated how DNA polymerase {beta} (Pol {beta}), a model mammalian polymerase, bypasses a templating Fapy*dG, inserts Fapy*dGTP, and extends from Fapy*dG at the primer terminus. When Fapy*dG is present in the template, Pol {beta} incorporates TMP less efficiently than either dCMP or dAMP. Kinetic analysis revealed that Fapy*dGTP is a poor substrate but is incorporated [~]3-times more efficiently opposite dA than dC. Extension from Fapy*dG at the 3-terminus of a nascent primer is inefficient due to the primer terminus being poorly positioned for catalysis. Together these data indicate that mutagenic bypass of Fapy*dG is likely to be the source of the mutagenic effects of the lesion and not Fapy*dGTP. These experiments increase our understanding of the promutagenic effects of Fapy*dG.

biochemistry↗

bioRxiv2023

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.

biochemistry↗

bioRxiv2023

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

Base excision repair is the main pathway involved in active DNA demethylation. 5-formylctyosine and 5-carboxylcytosine, two oxidized moieties of methylated cytosine, are recognized and removed by thymine DNA glycosylase (TDG) to generate an abasic site. Using single molecule fluorescence experiments, we studied TDG in the presence and absence of 5-formylctyosine. TDG exhibits multiple modes of linear diffusion, including hopping and sliding, in search of a lesion. We probed TDG active site variants and truncated N-terminus revealing how these variants alter the lesion search and recognition mechanism of TDG. On DNA containing an undamaged nucleosome, TDG was found to either bypass, colocalize with, or encounter but not bypass the nucleosome. However, truncating the N-terminus reduced the number of interactions with the nucleosome. Our findings provide unprecedented mechanistic insights into how TDG searches for DNA lesions in chromatin.

biophysics↗

bioRxiv2023

The VEGF G-quadruplex forming promoter is repaired via long-patch BER

In response to oxidative damage, base excision repair (BER) enzymes perturb the structural equilibrium of the VEGF promoter between B-form and G4 DNA conformations, resulting in epigenetic-like modifications of gene expression. However, the mechanistic details remain enigmatic, including the activity and coordination of BER enzymes on the damaged G4 promoter. To address this, we investigated the ability of each BER factor to conduct its repair activity on VEGF promoter G4 DNA substrates by employing pre-steady-state kinetics assays and in vitro coupled BER assays. OGG1 was able to initiate BER on double-stranded VEGF promoter G4 DNA substrates. Moreover, pre-steady-state kinetics revealed that compared to B-form DNA, APE1 repair activity on the G4 was decreased [~]2-fold and is the result of slower product release as opposed to inefficient strand cleavage. Interestingly, Pol {beta} performs multiple insertions on G4 substates via strand displacement DNA synthesis in contrast to a single insertion on B-form DNA. The multiple insertions inhibit ligation of the Pol {beta} products, and hence BER is not completed on the VEGF G4 promoter substrates through canonical short-patch BER. Instead, repair requires the long-patch BER flap-endonuclease activity of FEN1 in response to the multiple insertions by Pol {beta} prior to ligation. Because the BER proteins and their repair activities are a key part of the VEGF transcriptional enhancement in response to oxidative DNA damage of the G4 VEGF promoter, the new insights reported here on BER activity in the context of this promoter are relevant toward understanding the mechanism of transcriptional regulation.

biochemistry↗

bioRxiv2022

Visualizing the Coordination of APE1 and DNA Polymerase β During Base Excision Repair

Base Excision Repair (BER) is carried out by a series of DNA repair proteins that function in a step-by-step process to identify, remove, and replace DNA damage. As DNA damage is processed during BER, the DNA transitions through various intermediate states, called BER intermediates, which if left exposed can develop into double-strand DNA breaks and trigger programmed cell death signaling. Previous studies have proposed that in order to minimize exposure of the BER intermediates, each protein may remain bound to its product prior to the next protein binding. Thus, a short-lived complex consisting of the BER intermediate, the incoming enzyme, and the outgoing enzyme may form between each step of the BER pathway. The transfer of BER intermediates between enzymes, known as BER coordination, has yet to be directly visualized and the mechanistic details of the process remain unclear. Here, we utilize single-molecule total internal reflection fluorescence (TIRF) microscopy to investigate the mechanism of BER coordination between apurinic/apyrimidinic endonuclease 1 (APE1) and DNA polymerase {beta} (Pol {beta}). When preformed complexes comprised of APE1 and the incised AP-site product were subsequently bound by Pol {beta}, the Pol {beta} enzyme dissociated shortly after binding in a majority of the observations. In the events where Pol {beta} binding was followed by APE1 dissociation (i.e., DNA hand-off), Pol {beta} had remained bound for a longer period of time to allow disassociation of APE1. Our results indicate that, in the absence of other BER factors, transfer of the BER intermediate from APE1 to Pol {beta} during BER is dependent on the dissociation kinetics of APE1 and the duration that Pol {beta} remains bound near the APE1-5 nick complex. These findings provide insight into how APE1 and Pol {beta} coordinate the transfer of DNA within the BER pathway.

biochemistry↗

bioRxiv2022

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 determined 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 identified 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 identified 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.

biochemistry↗

bioRxiv2021

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.

biochemistry↗

bioRxiv2021

Processing of oxidatively damaged DNA dirty ends by APE1

Reactive oxygen species attack the structure of DNA, thus altering its base-pairing properties. Consequently, oxidative stress-associated DNA lesions are a major source of the mutation load that gives rise to cancer and other diseases. Base excision repair (BER) is the pathway primarily tasked with repairing DNA base damage, with apurinic/apyrimidinic endonuclease (APE1) having both AP-endonuclease and 3 to 5 exonuclease (exo) DNA cleavage functions. The lesion 8-oxo-7,8-dihydroguanine (8-oxoG) can enter the genome as either a product of direct damage to the DNA, or through polymerase insertion at the 3-end of a DNA strand during replication or repair. Importantly, 3-8-oxoG impairs the ligation step of BER and therefore must be removed by the exo activity of a surrogate enzyme to prevent double stranded breaks and cell death. In the present study, we characterize the exo activity of APE1 on 3-8-oxoG substrates. These structures demonstrate that APE1 uses a unified mechanism for its exo activities that differs from its more canonical AP-endonuclease activity. In addition, through complementation of the structural data with enzyme kinetics and binding studies employing both wild-type and rationally designed APE1 mutants, we were able to identify and characterize unique protein:DNA contacts that specifically mediate 8-oxoG removal by APE1.

biochemistry↗

bioRxiv2021

Construction of a 3-color prism-based TIRF microscope to study the interactions and dynamics of macromolecules

Single-molecule total internal reflection fluorescence (TIRF) microscopy allows for real-time visualization of macromolecular dynamics and complex assembly. Prism-based TIRF microscopes (prismTIRF) are relatively simple to operate and can be easily modulated to fit the needs of a wide variety of experimental applications. While building a prismTIRF microscope without expert assistance can pose a significant challenge, the components needed to build a prismTIRF microscope are relatively affordable and, with some guidance, the assembly can be completed by a determined novice. Here, we provide an easy-to-follow guide for the design, assembly, and operation of a 3-color prismTIRF microscope which can be utilized for the study macromolecular complexes, including the multi-component protein-DNA complexes responsible for DNA repair, replication, and transcription. Our hope is that this article can assist laboratories that aspire to implement single-molecule TIRF techniques, and consequently expand the application of this technology to a broader spectrum of scientific questions.

biochemistry↗