Antagonistic roles of canonical and Alternative-RPA in disease-associated tandem CAG repeat instability.

Expansions of repeat DNA tracts cause >70 diseases, and ongoing expansions in brains exacerbate disease. During expansion mutations, single-stranded DNAs (ssDNAs) form slipped-DNAs. We find the ssDNA-binding complexes canonical replication protein A (RPA1, RPA2, and RPA3) and Alternative-RPA (RPA1, RPA3, and primate-specific RPA4) are upregulated in Huntington disease and spinocerebellar ataxia type 1 (SCA1) patient brains. Protein interactomes of RPA and Alt-RPA reveal unique and shared partners, including modifiers of CAG instability and disease presentation. RPA enhances in vitro melting, FAN1 excision, and repair of slipped-CAGs and protects against CAG expansions in human cells. RPA overexpression in SCA1 mouse brains ablates expansions, coincident with decreased ATXN1 aggregation, reduced brain DNA damage, improved neuron morphology, and rescued motor phenotypes. In contrast, Alt-RPA inhibits melting, FAN1 excision, and repair of slipped-CAGs and promotes CAG expansions. These findings suggest a functional interplay between the two RPAs where Alt-RPA may antagonistically offset RPA's suppression of disease-associated repeat expansions, which may extend to other DNA processes.

Transient RNA-DNA Hybrids Are Required for Efficient Double-Strand Break Repair.

RNA-DNA hybrids are a major internal cause of DNA damage within cells, and their degradation by RNase H enzymes is important for maintaining genomic stability. Here, we identified an unexpected role for RNA-DNA hybrids and RNase H enzymes in DNA repair. Using a site-specific DNA double-strand break (DSB) system in Schizosaccharomyces pombe, we showed that RNA-DNA hybrids form as part of the homologous-recombination (HR)-mediated DSB repair process and that RNase H enzymes are essential for their degradation and efficient completion of DNA repair. Deleting RNase H stabilizes RNA-DNA hybrids around DSB sites and strongly impairs recruitment of the ssDNA-binding RPA complex. In contrast, overexpressing RNase H1 destabilizes these hybrids, leading to excessive strand resection and RPA recruitment and to severe loss of repeat regions around DSBs. Our study challenges the existing model of HR-mediated DSB repair and reveals a surprising role for RNA-DNA hybrids in maintaining genomic stability.

SUMOylation of HNRNPA2B1 modulates RPA dynamics during unperturbed replication and genotoxic stress responses.

Replication protein A (RPA) is a major regulator of eukaryotic DNA metabolism involved in multiple essential cellular processes. Maintaining appropriate RPA dynamics is crucial for cells to prevent RPA exhaustion, which can lead to replication fork breakage and replication catastrophe. However, how cells regulate RPA availability during unperturbed replication and in response to stress has not been well elucidated. Here, we show that HNRNPA2B1SUMO functions as an endogenous inhibitor of RPA during normal replication. HNRNPA2B1SUMO associates with RPA through recognizing the SUMO-interacting motif (SIM) of RPA to inhibit RPA accumulation at replication forks and impede local ATR activation. Declining HNRNPA2SUMO induced by DNA damage will release nuclear soluble RPA to localize to chromatin and enable ATR activation. Furthermore, we characterize that HNRNPA2B1 hinders homologous recombination (HR) repair via limiting RPA availability, thus conferring sensitivity to PARP inhibitors. These findings establish HNRNPA2B1 as a critical player in RPA-dependent surveillance networks.

A PARylation-phosphorylation cascade promotes TOPBP1 loading and RPA-RAD51 exchange in homologous recombination.

The efficiency of homologous recombination (HR) in the repair of DNA double-strand breaks (DSBs) is closely associated with genome stability and tumor response to chemotherapy. While many factors have been functionally characterized in HR, such as TOPBP1, their precise regulation remains unclear. Here, we report that TOPBP1 interacts with the RNA-binding protein HTATSF1 in a cell-cycle- and phosphorylation-dependent manner. Mechanistically, CK2 phosphorylates HTATSF1 to facilitate binding to TOPBP1, which promotes S-phase-specific TOPBP1 recruitment to damaged chromatin and subsequent RPA/RAD51-dependent HR, genome integrity, and cancer-cell viability. The localization of HTATSF1-TOPBP1 to DSBs is potentially independent of the transcription-coupled RNA-binding and processing capacity of HTATSF1 but rather relies on the recognition of poly(ADP-ribosyl)ated RPA by HTATSF1, which can be blunted with PARP inhibitors. Together, our study provides a mechanistic insight into TOPBP1 loading at HR-prone DSB sites via HTATSF1 and reveals how RPA-RAD51 exchange is tuned by a PARylation-phosphorylation cascade.

Replication protein A associates with nucleolar R loops and regulates rRNA transcription and nucleolar morphology.

The nucleolus is an important cellular compartment in which ribosomal RNAs (rRNAs) are transcribed and where certain stress pathways that are crucial for cell growth are coordinated. Here we report novel functions of the DNA replication and repair factor replication protein A (RPA) in control of nucleolar homeostasis. We show that loss of the DNA:RNA helicase senataxin (SETX) promotes RPA nucleolar localization, and that this relocalization is dependent on the presence of R loops. Notably, this nucleolar RPA phenotype was also observed in the presence of camptothecin (CPT)-induced genotoxic stress, as well as in SETX-deficient AOA2 patient fibroblasts. Extending these results, we found that RPA is recruited to rDNA following CPT treatment, where RPA prevents R-loop-induced DNA double-strand breaks. Furthermore, we show that loss of RPA significantly decreased 47S pre-rRNA levels, which was accompanied by increased expression of both RNAP II-mediated "promoter and pre-rRNA antisense" RNA as well as RNAP I-transcribed intragenic spacer RNAs. Finally, and likely reflecting the above, we found that loss of RPA promoted nucleolar structural disorganization, characterized by the appearance of reduced size nucleoli. Our findings both indicate new roles for RPA in nucleoli through pre-rRNA transcriptional control and also emphasize that RPA function in nucleolar homeostasis is linked to R-loop resolution under both physiological and pathological conditions.

Processing of DNA Polymerase-Blocking Lesions during Genome Replication Is Spatially and Temporally Segregated from Replication Forks.

Tracing DNA repair factors by fluorescence microscopy provides valuable information about how DNA damage processing is orchestrated within cells. Most repair pathways involve single-stranded DNA (ssDNA), making replication protein A (RPA) a hallmark of DNA damage and replication stress. RPA foci emerging during S phase in response to tolerable loads of polymerase-blocking lesions are generally thought to indicate stalled replication intermediates. We now report that in budding yeast they predominantly form far away from sites of ongoing replication, and they do not overlap with any of the repair centers associated with collapsed replication forks or double-strand breaks. Instead, they represent sites of postreplicative DNA damage bypass involving translesion synthesis and homologous recombination. We propose that most RPA and recombination foci induced by polymerase-blocking lesions in the replication template are clusters of repair tracts arising from replication centers by polymerase re-priming and subsequent expansion of daughter-strand gaps over the course of S phase.

The Configuration of RPA, RAD51, and DMC1 Binding in Meiosis Reveals the Nature of Critical Recombination Intermediates.

Meiotic recombination proceeds via binding of RPA, RAD51, and DMC1 to single-stranded DNA (ssDNA) substrates created after formation of programmed DNA double-strand breaks. Here we report high-resolution in vivo maps of RPA and RAD51 in meiosis, mapping their binding locations and lifespans to individual homologous chromosomes using a genetically engineered hybrid mouse. Together with high-resolution microscopy and DMC1 binding maps, we show that DMC1 and RAD51 have distinct spatial localization on ssDNA: DMC1 binds near the break site, and RAD51 binds away from it. We characterize inter-homolog recombination intermediates bound by RPA in vivo, with properties expected for the critical displacement loop (D-loop) intermediates. These data support the hypothesis that DMC1, not RAD51, performs strand exchange in mammalian meiosis. RPA-bound D-loops can be resolved as crossovers or non-crossovers, but crossover-destined D-loops may have longer lifespans. D-loops resemble crossover gene conversions in size, but their extent is similar in both repair pathways.

RPA Phosphorylation Inhibits DNA Resection.

Genetic recombination in all kingdoms of life initiates when helicases and nucleases process (resect) the free DNA ends to expose single-stranded DNA (ssDNA) overhangs. Resection regulation in bacteria is programmed by a DNA sequence, but a general mechanism limiting resection in eukaryotes has remained elusive. Using single-molecule imaging of reconstituted human DNA repair factors, we identify phosphorylated RPA (pRPA) as a negative resection regulator. Bloom's syndrome (BLM) helicase together with exonuclease 1 (EXO1) and DNA2 nucleases catalyze kilobase-length DNA resection on nucleosome-coated DNA. The resulting ssDNA is rapidly bound by RPA, which further stimulates DNA resection. RPA is phosphorylated during resection as part of the DNA damage response (DDR). Remarkably, pRPA inhibits DNA resection in cellular assays and in vitro via inhibition of BLM helicase. pRPA suppresses BLM initiation at DNA ends and promotes the intrinsic helicase strand-switching activity. These findings establish that pRPA provides a feedback loop between DNA resection and the DDR.

Rtt105 stimulates Rad51-ssDNA assembly and orchestrates Rad51 and RPA actions to promote homologous recombination repair.

Homologous recombination (HR) is essential for the maintenance of genome stability. During HR, Replication Protein A (RPA) rapidly coats the 3'-tailed single-strand DNA (ssDNA) generated by end resection. Then, the ssDNA-bound RPA must be timely replaced by Rad51 recombinase to form Rad51 nucleoprotein filaments that drive homology search and HR repair. How cells regulate Rad51 assembly dynamics and coordinate RPA and Rad51 actions to ensure proper HR remains poorly understood. Here, we identified that Rtt105, a Ty1 transposon regulator, acts to stimulate Rad51 assembly and orchestrate RPA and Rad51 actions during HR. We found that Rtt105 interacts with Rad51 in vitro and in vivo and restrains the adenosine 5' triphosphate (ATP) hydrolysis activity of Rad51. We showed that Rtt105 directly stimulates dynamic Rad51-ssDNA assembly, strand exchange, and D-loop formation in vitro. Notably, we found that Rtt105 physically regulates the binding of Rad51 and RPA to ssDNA via different motifs and that both regulations are necessary and epistatic in promoting Rad51 nucleation, strand exchange, and HR repair. Consequently, disrupting either of the interactions impaired HR and conferred DNA damage sensitivity, underscoring the importance of Rtt105 in orchestrating the actions of Rad51 and RPA. Our work reveals additional layers of mechanisms regulating Rad51 filament dynamics and the coordination of HR.

PACRAT: pathogen detection with aptamer-observed cascaded recombinase polymerase amplification-in vitro transcription.

The SARS-CoV-2 pandemic underscored the need for early, rapid, and widespread pathogen detection tests that are readily accessible. Many existing rapid isothermal detection methods use the recombinase polymerase amplification (RPA), which exhibits polymerase chain reaction (PCR)-like sensitivity, specificity, and even higher speed. However, coupling RPA to other enzymatic reactions has proven difficult. For the first time, we demonstrate that with tuning of buffer conditions and optimization of reagent concentrations, RPA can be cascaded into an in vitro transcription reaction, enabling detection using fluorescent aptamers in a one-pot reaction. We show that this reaction, which we term PACRAT (pathogen detection with aptamer-observed cascaded recombinase polymerase amplification-in vitro transcription) can be used to detect SARS-CoV-2 RNA with single-copy detection limits, Escherichia coli with single-cell detection limits, and 10-min detection times. Further demonstrating the utility of our one-pot, cascaded amplification system, we show PACRAT can be used for multiplexed detection of the pathogens SARS-CoV-2 and E. coli, along with multiplexed detection of two variants of SARS-CoV-2.

The functional significance of the RPA- and PCNA-dependent recruitment of Pif1 to DNA.

Pif1 family helicases are multifunctional proteins conserved in eukaryotes, from yeast to humans. They are important for the genome maintenance in both nuclei and mitochondria, where they have been implicated in Okazaki fragment processing, replication fork progression and termination, telomerase regulation and DNA repair. While the Pif1 helicase activity is readily detectable on naked nucleic acids in vitro, the in vivo functions rely on recruitment to DNA. We identify the single-stranded DNA binding protein complex RPA as the major recruiter of Pif1 in budding yeast, in addition to the previously reported Pif1-PCNA interaction. The two modes of the Pif1 recruitment act independently during telomerase inhibition, as the mutations in the Pif1 motifs disrupting either of the recruitment pathways act additively. In contrast, both recruitment mechanisms are essential for the replication-related roles of Pif1 at conventional forks and during the repair by break-induced replication. We propose a molecular model where RPA and PCNA provide a double anchoring of Pif1 at replication forks, which is essential for the Pif1 functions related to the fork movement.

Genome-wide Identification of Structure-Forming Repeats as Principal Sites of Fork Collapse upon ATR Inhibition.

DNA polymerase stalling activates the ATR checkpoint kinase, which in turn suppresses fork collapse and breakage. Herein, we describe use of ATR inhibition (ATRi) as a means to identify genomic sites of problematic DNA replication in murine and human cells. Over 500 high-resolution ATR-dependent sites were ascertained using two distinct methods: replication protein A (RPA)-chromatin immunoprecipitation (ChIP) and breaks identified by TdT labeling (BrITL). The genomic feature most strongly associated with ATR dependence was repetitive DNA that exhibited high structure-forming potential. Repeats most reliant on ATR for stability included structure-forming microsatellites, inverted retroelement repeats, and quasi-palindromic AT-rich repeats. Notably, these distinct categories of repeats differed in the structures they formed and their ability to stimulate RPA accumulation and breakage, implying that the causes and character of replication fork collapse under ATR inhibition can vary in a DNA-structure-specific manner. Collectively, these studies identify key sources of endogenous replication stress that rely on ATR for stability.

"Helicase" Activity promoted through dynamic interactions between a ssDNA translocase and a diffusing SSB protein.

Replication protein A (RPA) is a eukaryotic single-stranded (ss) DNA-binding (SSB) protein that is essential for all aspects of genome maintenance. RPA binds ssDNA with high affinity but can also diffuse along ssDNA. By itself, RPA is capable of transiently disrupting short regions of duplex DNA by diffusing from a ssDNA that flanks the duplex DNA. Using single-molecule total internal reflection fluorescence and optical trapping combined with fluorescence approaches, we show that S. cerevisiae Pif1 can use its ATP-dependent 5' to 3' translocase activity to chemomechanically push a single human RPA (hRPA) heterotrimer directionally along ssDNA at rates comparable to those of Pif1 translocation alone. We further show that using its translocation activity, Pif1 can push hRPA from a ssDNA loading site into a duplex DNA causing stable disruption of at least 9 bp of duplex DNA. These results highlight the dynamic nature of hRPA enabling it to be readily reorganized even when bound tightly to ssDNA and demonstrate a mechanism by which directional DNA unwinding can be achieved through the combined action of a ssDNA translocase that pushes an SSB protein. These results highlight the two basic requirements for any processive DNA helicase: transient DNA base pair melting (supplied by hRPA) and ATP-dependent directional ssDNA translocation (supplied by Pif1) and that these functions can be unlinked by using two separate proteins.

HEI10 is subject to phase separation and mediates RPA1a degradation during meiotic interference-sensitive crossover formation.

Reciprocal exchanges of DNA between homologous chromosomes during meiosis, or crossovers (COs), shuffle genetic information in gametes and progeny. In many eukaryotes, the majority of COs (class I COs) are sensitive to a phenomenon called interference, which influences the occurrence of closely spaced double COs. Class I COs depend on a group of factors called ZMM (Zip, Msh, Mer) proteins including HEI10 (Human Enhancer of Invasion-10). However, how these proteins are recruited to class I CO sites is unclear. Here, we show that HEI10 forms foci on chromatin via a liquid-liquid phase separation (LLPS) mechanism that relies on residue Ser70. A HEI10S70F allele results in LLPS failure and a defect in class I CO formation. We further used immunoprecipitation-mass spectrometry to identify RPA1a (Replication Protein A 1) as a HEI10 interacting protein. Surprisingly, we find that RPA1a also undergoes phase separation and its ubiquitination and degradation are directly regulated by HEI10. We also show that HEI10 is required for the condensation of other class I CO factors. Thus, our results provide mechanistic insight into how meiotic class I CO formation is controlled by HEI10 coupling LLPS and ubiquitination.

The RPA-RNF20-SNF2H cascade promotes proper chromosome segregation and homologous recombination repair.

The human tumor suppressor Ring finger protein 20 (RNF20)-mediated histone H2B monoubiquitination (H2Bub) is essential for proper chromosome segregation and DNA repair. However, what is the precise function and mechanism of RNF20-H2Bub in chromosome segregation and how this pathway is activated to preserve genome stability remain unknown. Here, we show that the single-strand DNA-binding factor Replication protein A (RPA) interacts with RNF20 mainly in the S and G2/M phases and recruits RNF20 to mitotic centromeres in a centromeric R-loop-dependent manner. In parallel, RPA recruits RNF20 to chromosomal breaks upon DNA damage. Disruption of the RPA-RNF20 interaction or depletion of RNF20 increases mitotic lagging chromosomes and chromosome bridges and impairs BRCA1 and RAD51 loading and homologous recombination repair, leading to elevated chromosome breaks, genome instability, and sensitivities to DNA-damaging agents. Mechanistically, the RPA-RNF20 pathway promotes local H2Bub, H3K4 dimethylation, and subsequent SNF2H recruitment, ensuring proper Aurora B kinase activation at centromeres and efficient loading of repair proteins at DNA breaks. Thus, the RPA-RNF20-SNF2H cascade plays a broad role in preserving genome stability by coupling H2Bub to chromosome segregation and DNA repair.

NHP2 downregulation counteracts hTR-mediated activation of the DNA damage response at ALT telomeres.

About 10% of cancer cells employ the "alternative lengthening of telomeres" (ALT) pathway instead of re-activating the hTERT subunit of human telomerase. The hTR RNA subunit is also abnormally silenced in some ALT+ cells not expressing hTERT, suggesting a possible negative non-canonical impact of hTR on ALT. Indeed, we show that ectopically expressed hTR reduces phosphorylation of ssDNA-binding protein RPA (p-RPAS33 ) at ALT telomeres by promoting the hnRNPA1- and DNA-PK-dependent depletion of RPA. The resulting defective ATR checkpoint signaling at telomeres impairs recruitment of the homologous recombination protein, RAD51. This induces ALT telomere fragility, increases POLD3-dependent C-circle production, and promotes the recruitment of the DNA damage marker 53BP1. In ALT+ cells that naturally retain hTR expression, NHP2 H/ACA ribonucleoprotein levels are downregulated, likely in order to restrain DNA damage response (DDR) activation at telomeres through reduced 53BP1 recruitment. This unexpected role of NHP2 is independent from hTR's non-canonical function in modulating telomeric p-RPAS33 . Collectively, our study shines new light on the interference between telomerase- and ALT-dependent pathways and unravels a crucial role for hTR and NHP2 in DDR regulation at ALT telomeres.

PrimPol-mediated repriming facilitates replication traverse of DNA interstrand crosslinks.

DNA interstrand crosslinks (ICLs) induced by endogenous aldehydes or chemotherapeutic agents interfere with essential processes such as replication and transcription. ICL recognition and repair by the Fanconi Anemia pathway require the formation of an X-shaped DNA structure that may arise from convergence of two replication forks at the crosslink or traversing of the lesion by a single replication fork. Here, we report that ICL traverse strictly requires DNA repriming events downstream of the lesion, which are carried out by PrimPol, the second primase-polymerase identified in mammalian cells after Polα/Primase. The recruitment of PrimPol to the vicinity of ICLs depends on its interaction with RPA, but not on FANCM translocase or the BLM/TOP3A/RMI1-2 (BTR) complex that also participate in ICL traverse. Genetic ablation of PRIMPOL makes cells more dependent on the fork convergence mechanism to initiate ICL repair, and PRIMPOL KO cells and mice display hypersensitivity to ICL-inducing drugs. These results open the possibility of targeting PrimPol activity to enhance the efficacy of chemotherapy based on DNA crosslinking agents.

Human Bocavirus 1 NP1 acts as an ssDNA-binding protein to help AAV2 DNA replication and cooperates with RPA to regulate AAV2 capsid expression.

Adeno-associated virus (AAV) requires co-infection with helper virus for efficient replication. We previously reported that Human Bocavirus 1 (HBoV1) genes, including NP1, NS2, and BocaSR, were critical for AAV2 replication. Here, we first demonstrate the essential roles of the NP1 protein in AAV2 DNA replication and protein expression. We show that NP1 binds to single-strand DNA (ssDNA) at least 30 nucleotides (nt) in length in a sequence-independent manner. Furthermore, NP1 colocalized with the BrdU-labeled AAV2 DNA replication center, and the loss of the ssDNA-binding ability of NP1 by site-directed mutation completely abolished AAV2 DNA replication. We used affinity-tagged NP1 protein to identify host cellular proteins associated with NP1 in cells cotransfected with the HBoV1 helper genes and AAV2 duplex genome. Of the identified proteins, we demonstrate that NP1 directly binds to the DBD-F domain of the RPA70 subunit with a high affinity through the residues 101-121. By reconstituting the heterotrimer protein RPA in vitro using gel filtration, we demonstrate that NP1 physically associates with RPA to form a heterologous complex characterized by typical fast-on/fast-off kinetics. Following a dominant-negative strategy, we found that NP1-RPA complex mainly plays a role in expressing AAV2 capsid protein by enhancing the transcriptional activity of the p40 promoter. Our study revealed a novel mechanism by which HBoV1 NP1 protein supports AAV2 DNA replication and capsid protein expression through its ssDNA-binding ability and direct interaction with RPA, respectively.IMPORTANCERecombinant adeno-associated virus (rAAV) vectors have been extensively used in clinical gene therapy strategies. However, a limitation of these gene therapy strategies is the efficient production of the required vectors, as AAV alone is replication-deficient in the host cells. HBoV1 provides the simplest AAV2 helper genes consisting of NP1, NS2, and BocaSR. An important question regarding the helper function of HBoV1 is whether it provides any direct function that supports AAV2 DNA replication and protein expression. Also of interest is how HBoV1 interplays with potential host factors to constitute a permissive environment for AAV2 replication. Our studies revealed that the multifunctional protein NP1 plays important roles in AAV2 DNA replication via its sequence-independent ssDNA-binding ability and in regulating AAV2 capsid protein expression by physically interacting with host protein RPA. Our findings present theoretical guidance for the future application of the HBoV1 helper genes in the rAAV vector production.

Fluorescent human RPA to track assembly dynamics on DNA.

DNA metabolic processes including replication, repair, recombination, and telomere maintenance occur on single-stranded DNA (ssDNA). In each of these complex processes, dozens of proteins function together on the ssDNA template. However, when double-stranded DNA is unwound, the transiently open ssDNA is protected and coated by the high affinity heterotrimeric ssDNA binding Replication Protein A (RPA). Almost all downstream DNA processes must first remodel/remove RPA or function alongside to access the ssDNA occluded under RPA. Formation of RPA-ssDNA complexes trigger the DNA damage checkpoint response and is a key step in activating most DNA repair and recombination pathways. Thus, in addition to protecting the exposed ssDNA, RPA functions as a gatekeeper to define functional specificity in DNA maintenance and genomic integrity. RPA achieves functional dexterity through a multi-domain architecture utilizing several DNA binding and protein-interaction domains connected by flexible linkers. This flexible and modular architecture enables RPA to adopt a myriad of configurations tailored for specific DNA metabolic roles. To experimentally capture the dynamics of the domains of RPA upon binding to ssDNA and interacting proteins we here describe the generation of active site-specific fluorescent versions of human RPA (RPA) using 4-azido-L-phenylalanine (4AZP) incorporation and click chemistry. This approach can also be applied to site-specific modifications of other multi-domain proteins. Fluorescence-enhancement through non-canonical amino acids (FEncAA) and Förster Resonance Energy Transfer (FRET) assays for measuring dynamics of RPA on DNA are also described. The fluorescent human RPA described here will enable high-resolution structure-function analysis of RPA-ssDNA interactions.

Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide excision repair.

Nucleotide excision repair (NER) promotes genomic integrity by removing bulky DNA adducts introduced by external factors such as ultraviolet light. Defects in NER enzymes are associated with pathological conditions such as Xeroderma Pigmentosum, trichothiodystrophy, and Cockayne syndrome. A critical step in NER is the binding of the Xeroderma Pigmentosum group A protein (XPA) to the ss/ds DNA junction. To better capture the dynamics of XPA interactions with DNA during NER we have utilized the fluorescence enhancement through non-canonical amino acids (FEncAA) approach. 4-azido-L-phenylalanine (4AZP or pAzF) was incorporated at Arg-158 in human XPA and conjugated to Cy3 using strain-promoted azide-alkyne cycloaddition. The resulting fluorescent XPA protein (XPACy3) shows no loss in DNA binding activity and generates a robust change in fluorescence upon binding to DNA. Here we describe methods to generate XPACy3 and detail in vitro experimental conditions required to stably maintain the protein during biochemical and biophysical studies.