BMB Reports 2025; 58(1): 17-23  https://doi.org/10.5483/BMBRep.2024-0178
Single-molecule studies of repair proteins in base excision repair
Donghun Lee & Gwangrog Lee*
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, Korea
Correspondence to: Tel: +82-42-350-2614; Fax: +82-42-350-5614; E-mail: ifglee@kaist.ac.kr
Received: October 21, 2024; Revised: December 7, 2024; Accepted: December 7, 2024; Published online: January 14, 2025.
© Korean Society for Biochemistry and Molecular Biology. All rights reserved.

cc This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Base excision repair (BER) is an essential cellular mechanism that repairs small, non-helix-distorting base lesions in DNA, resulting from oxidative damage, alkylation, deamination, or hydrolysis. This review highlights recent advances in understanding the molecular mechanisms of BER enzymes through single-molecule studies. We discuss the roles of DNA glycosylases in lesion recognition and excision, with a focus on facilitated diffusion mechanisms such as sliding and hopping that enable efficient genome scanning. The dynamics of apurinic/apyrimidinic endonucleases, especially the coordination between APE1 and DNA polymerase β (Pol β), are explored to demonstrate their crucial roles in processing abasic sites. The review further explores the short-patch and long-patch BER pathways, emphasizing the activities of Pol β, XRCC1, PARP1, FEN1, and PCNA in supporting repair synthesis and ligation. Additionally, we highlight the emerging role of UV-DDB as a general damage sensor in BER, extending its recognized function beyond nucleotide excision repair. Single-molecule techniques have been instrumental in uncovering the complex interactions and mechanisms of BER proteins, offering unprecedented insights that could guide future therapeutic strategies for maintaining genomic stability.
Keywords: Base excision repair, DNA damage, Protein-DNA interactions, Repair proteins, Single-molecule
INTRODUCTION

Base excision repair (BER) is a critical cellular mechanism that repairs small, non-helix-distorting base lesions in DNA caused by oxidative damage, alkylation, deamination, or spontaneous hydrolysis (1-4). BER is initiated by DNA glycosylases which identify and remove damaged bases, creating an apurinic/apyrimidinic site (AP site) (5, 6) (Fig. 1). Apurinic/apyrimidinic endonucleases then cleave the DNA backbone at the AP site, generating a single-strand break (7, 8) (Fig. 1). Depending on the type of repair, the pathway proceeds via either the short-patch or long-patch BER mechanism, recruiting specific enzymes (9, 10) (Fig. 1). In the short-patch pathway, DNA polymerase β (Pol β), XRCC1, PARP1, and DNA ligase III (LIG3) primarily repair a single nucleotide (Fig. 1) (11, 12). In long-patch BER, DNA polymerase δ/ε (Pol δ/ε), PCNA, FEN1, and DNA ligase I (LIG1) restore several nucleotides (Fig. 1) (13-15).

Given the complexity of BER, precise detection techniques are essential for understanding the dynamics and interactions of the repair proteins. Single-molecule studies have proven to be powerful tools for dissecting the molecular mechanisms of BER with an unprecedented level of detail. Techniques such as single-molecule Förster resonance energy transfer (smFRET), single-molecule flow-stretching (smFS), optical tweezers, and the single-molecule DNA tightrope assay have offered new insights into how BER proteins recognize and process DNA lesions (16-19) (Fig. 2). These methods allow researchers to observe the real-time interactions of individual proteins with DNA, unveiling their target search mechanisms, binding dynamics, conformational changes, and target recognition (20-25). These single-molecule techniques have provided valuable insights into the spatiotemporal dynamics of BER, enhancing our understanding of how repair proteins orchestrate the DNA repair process and identifying potential new targets for therapeutic intervention, particularly in cancer (26).

DNA GLYCOSYLASES IN BER

In the BER pathway, DNA glycosylase serves as the initiating enzyme that interrogates and removes damaged bases to preserve genomic integrity. Both bacteria and humans possess various DNA glycosylases, each specifically evolved to detect and repair types of base damage (27) (Table 1). A major challenge for all DNA repair proteins is the efficient scanning of the vast genome to locate rare lesions. Single-molecule studies have significantly advanced our understanding of how glycosylases address this challenge by employing facilitated diffusion. Facilitated diffusion enhances search efficiency by combining one-dimensional (1D) diffusion along the DNA with three-dimensional (3D) diffusion in solution (28). Sliding involves the enzyme moving along the DNA while maintaining continuous contact with the phosphate backbone, and hopping involves the enzyme transiently dissociating from the DNA and re-associating nearby without fully diffusing into the bulk solution (29). To distinguish between sliding and hopping, researchers use methods such as NaCl titration and roadblock assays (30, 31). Sliding is less influenced by ionic strength because it depends on continuous association between the protein and DNA, leading to a 1D diffusion constant that is relatively independent of salt concentration. In contrast, hopping occurs with transient dissociation and re-association of the protein with the DNA and is sensitive to salt concentration, which influences electrostatic interactions. As salt concentration increases, these interactions weaken, diminishing the DNA and protein interaction affinity. This reduction in affinity elevates the likelihood of the enzyme detaching from the DNA during 1D translocation events, but when bound to DNA, the enzyme can diffuse more freely, thus enhancing the 1D diffusion constant. Consequently, the salt dependence of the 1D diffusion constant helps differentiate between sliding and hopping mechanisms of proteins that interact electrostatically with DNA (29). Roadblock analysis differentiates between hopping and sliding mechanisms by utilizing proteins that bind to specific DNA sites along the DNA as physical barriers (30). If the searching glycosylase cannot overcome these roadblocks, it likely employs a sliding mechanism; if it can, then hopping is probably involved.

A single-molecule study on oxoguanine DNA glycosylase 1 (OGG1) has investigated its target-searching mechanisms (32). In these experiments, lambda DNA is stretched and aligned by shear flow, and Cy3- labeled OGG1 is observed moving along the DNA. OGG1 was found to move rapidly along DNA through a sliding mechanism that maintains persistent contact with the DNA helix. The research suggests that this near-barrierless sliding enables OGG1 to scan DNA effectively and selectively excise damaged bases, thereby preventing mutations caused by oxidative stress. Further analysis of OGG1 diffusion on DNA reveals a two-state kinetic model that characterizes OGG1’s movement along DNA, alternating between loosely bound and tightly bound states (33). The loosely bound state allows the protein to slide rapidly along the DNA, while the tightly bound yet mobile state facilitates more precise detection of oxidative DNA damage. This dual-state mechanism ensures a balance between rapid scanning and accurate recognition by OGG1, optimizing its role in DNA repair. The application of the Single-Molecule Analysis of DNA-binding proteins from Nuclear Extracts (SMADNE) technique demonstrated significant differences in OGG1’s binding dynamics between purified proteins and nuclear extracts (34). This observation is crucial as it shows that OGG1’s behavior alters in the more complex, physiologically relevant environment of nuclear extracts, which include various nuclear proteins possibly influencing OGG1’s binding and search for damaged DNA. In subsequent studies, the authors offered additional insights by comparing purified OGG1 directly with OGG1 in nuclear extracts. They observed that purified OGG1 showed longer binding times on undamaged DNA than OGG1 in nuclear extracts (35). This indicates that the presence of other proteins and factors in nuclear extracts can modify the enzyme’s search and binding behavior. Furthermore, OGG1 exhibits different binding dynamics in nuclear extracts, likely due to interactions with other proteins that stabilize its attachment to oxidative DNA damage. This underscores the influence of the cellular environment on enzyme activity (34, 35). Overall, these findings emphasize the diverse mechanisms employed by glycosylases in target searching and binding, highlighting their adaptability and the effectiveness of the BER pathway in maintaining genomic stability.

Variations and combinations in target search strategies and binding modes are evident among different glycosylases. For instance, MUTYH glycosylase, which removes adenines mispaired with 8-oxoG, does not solely rely on a wedge residue for lesion recognition but utilizes it to stabilize the lesion-bound complex. Single-molecule fluorescence microscopy has shown that although wild-type MUTYH effectively locates and stably binds damage sites, wedge mutant variants exhibit decreased binding stability (36). Furthermore, AlkD glycosylase employs a distinctive non-base-flipping mechanism; it scans DNA without forming stable interrogation complexes, contrasting the common base-flipping strategy employed by other glycosylases (37). Various glycosylases have been studied using single-molecule techniques, providing dynamic insights into the diffusion mechanisms and binding modes of DNA glycosylases. These methods allow direct observations of individual protein movements and interactions with DNA, providing exceptional insights into how glycosylases efficiently locate and recognize damaged bases among a plethora of normal nucleotides. Extending such studies to other DNA repair proteins provides a significant opportunity to discover fundamental molecular mechanisms underlying DNA repair processes. This detailed understanding could lead to advancements in DNA repair research and aid in developing new strategies for maintaining genomic stability.

APURINIC/APYRIMIDINIC ENDONUCLEASES

When DNA glycosylase removes the damaged base, an AP site remains. The AP site is subsequently recognized by AP endonuclease, which cleaves the DNA backbone of the AP site to facilitate further repair (38-40). AP sites are one of the most frequently occurring lesion types in the genome, accumulating more than 10,000 such sites in a single human cell every day (41, 42). AP sites are highly cytotoxic and carcinogenic because they can lead to mutations during DNA replication and also form DNA-protein crosslinks (43). Given the importance of AP site processing, extensive research has been conducted to understand the mechanisms of AP endonucleases. A recent smFRET study focusing on Exonuclease III, a bacterial AP endonuclease, revealed an AP site anchor-based mechanism by which the intrinsically distributive enzyme becomes processive due to its strong binding affinity to the AP site, rapidly generating a transient ssDNA loop associated with the gap (22). Once the 3’ end releases from the AP endonuclease, polymerase I promptly initiates DNA synthesis and fills the gap. They also identified key determinants of functional selection between the two activities (i.e., AP endonuclease and exonuclease) (23). An aromatic residue, either W212 or F213, crucially recognizes the AP site and enhances AP endonuclease activity, with the F213 residue further stabilizing the melted state of the 3’ terminal nucleobases, leading to the catalytically competent state that activates the 3’→5’ exonuclease activity.

In human AP endonuclease (APE1), it performs a similar function by incising the DNA backbone at AP sites, generating a single-strand break with a 3’ hydroxyl and 5’ deoxyribose phosphate (dRP) termini (40). APE1 not only cleaves DNA at AP sites but also interacts with other BER proteins such as Pol β (44). This interaction is crucial for gap filling during the repair process, as APE1’s incision enables Pol β to synthesize new nucleotides, replacing the excised damaged bases. The structural and biochemical interactions between APE1 and Pol β ensure that the gap created at the AP site is accurately and efficiently filled .

In eukaryotic proteins, single-molecule studies have revealed insights into how BER coordination occurs between APE1 and Pol β (45). These studies found that when pre-formed complexes of APE1 and its product (which also acts as the substrate for Pol β) were bound by Pol β, the Pol β enzyme rapidly dissociated in most instances. However, in instances where Pol β binding was followed by APE1 dissociation, Pol β remained bound longer, facilitating APE1’s release. These results indicate that the transfer of the BER intermediate from APE1 to Pol β depends on the dissociation rate of APE1 and the duration the ternary complex remains intact on the abasic site. While Pol β is crucial for gap filling during the repair process, its lack of a proofreading function results in a relatively high error rate, potentially leading to the incorporation of incorrect nucleotides at the 3’ end. APE1 plays a vital role in removing these 3’ mismatches, thereby enhancing the fidelity of DNA repair (46, 47). Elucidating the mechanisms of coordination between APE1 and Pol β through single-molecule studies is highly beneficial. Understanding this interaction at the molecular level could provide deeper insights into DNA repair fidelity and the prevention of mutagenesis.

SHORT-PATCH BER

In eukaryotes, the short-patch BER pathway is characterized by the repair of a single nucleotide at the damaged site. Following the cleavage of the AP site by APE1, Pol β plays a central role with two key enzymatic activities: DNA synthesis (gap-filling) and AP lyase activity. Specifically, Pol β adds a single nucleotide to the 3’-hydroxyl end of the DNA strand and subsequently removes the 5’ deoxyribose phosphate (5’-dRP) group, which forms the base-free backbone. The coordination of AP lyase and DNA synthesis activities ensures that the repair process proceeds smoothly without accumulating intermediate lesions that could be cytotoxic or mutagenic. Structural studies indicate that Pol β undergoes conformational changes which facilitate the sequential execution of its dual functions (48).

XRCC1 (X-ray repair cross-complementing protein 1) functions as a scaffold protein, coordinating the interactions among various BER proteins (49). XRCC1 binds to both Pol β and DNA ligase III (LIG3), thereby facilitating the final ligation step after gap filling. LIG3 completes the repair process by sealing the nick in the DNA backbone. Poly (ADP-ribose) polymerase 1 (PARP1) is pivotal in sensing DNA damage, recognizing single-strand breaks, and acting as a mediator of repair by attracting XRCC1 to the damage site (50). When PARP1 binds to a DNA break, it triggers the DNA damage repair process, assembles and organizes the necessary repair factors, including XRCC1 and LIG3, and ensures the BER process is efficiently carried out. Single-molecule techniques such as SMADNE have been employed to examine PARP1’s interactions with DNA at the individual molecule level in the BER pathway (34). Furthermore, various single-molecule techniques have shown that PARP1 binds to DNA nicks and can condense undamaged DNA by stabilizing loops, a process influenced by mechanical forces such as tension applied to the DNA (51). Another study using single-molecule fluorescence microscopy demonstrated that PARP1 primarily searches for damage through three-dimensional diffusion but can switch to one-dimensional diffusion along the DNA following auto- poly(ADP-ribose)ylation (PARylation) or in the presence of APE1 (52). This transition enhances PARP1’s efficiency in locating and processing DNA lesions. Moreover, the dynamics of PARP1 binding and retention on DNA lesions, as demonstrated using smFRET, are modulated by PARP inhibitors (53). These inhibitors have varied effects on whether PARP1 remains bound to DNA or dissociates, potentially affecting the repair process. Understanding these interactions is vital, as the retention of PARP1 at damage sites can influence the recruitment of other repair proteins and the overall efficiency of BER. After APE1 cleaves the AP site, the precise sequential order in which Pol β, XRCC1, and PARP1 are recruited is crucial for the efficiency and accuracy of the BER pathway. Elucidating the exact sequence of these events is essential for understanding the coordination within the BER pathway. Single-molecule studies offer valuable insights into this process by directly observing the dynamics and interactions of these proteins at the single-molecule level, potentially revealing the temporal order and mechanisms of their recruitment following AP site incision.

LONG-PATCH BER

In contrast to short-patch BER, the long-patch repair pathway repairs 2 to 12 nucleotides (13). This pathway is typically initiated when the 5’-dRP terminus generated by APE1 resists removal by Pol β, necessitating the recruitment of additional proteins for complete repair. Polymerases δ and ε (Pol δ/ε) play critical roles in long-patch BER by synthesizing a DNA stretch beyond the initial nucleotide gap. This elongated synthesis displaces part of the damaged strand, forming a flap of several nucleotides. Flap endonuclease 1 (FEN1) then cleaves this 5’-flap structure, ensuring efficient removal of the displaced DNA (54). FEN1’s role is critical in preventing the accumulation of excess single-stranded DNA flaps, which can compromise repair fidelity. Recent single-molecule studies have offered substantial insights into the mechanisms by which FEN1 recognizes and cleaves flaps. Using smFRET, researchers demonstrated that FEN1 undergoes a sequential, multistep substrate interrogation process (55). This process involves threading the 5’ flap through a conserved helical gateway and bending the DNA at the base of the junction, allowing FEN1 to recognize specific substrates and restrict DNA bending to a common two-nucleotide unpairing intermediate step in 5’ nucleases, thus preventing nonspecific cleavage. Further smFRET analyses showed that FEN1 uses an induced-fit mechanism for substrate selectivity (56). FEN1 interacts with the DNA substrate via diffusion-limited kinetics, prompting a structural change that secures both enzyme and DNA in a stable, specific configuration. This coordination enhances FEN1’s ability to precisely recognize the correct DNA substrate, significantly reducing the risk of off-target cleavage and ensuring high accuracy in its endonuclease activity. Proliferating Cell Nuclear Antigen (PCNA) serves as a processivity factor for Pol δ/ε, improving the efficiency of long-patch repair (14). PCNA forms a clamp around the DNA, enabling Pol δ/ε to synthesize extensive DNA stretches without detaching. After the flap is cleaved, LIG1 seals the nick, completing the long-patch repair. This pathway is crucial for addressing complex lesions requiring more extensive repair than the single-nucleotide excision observed in short-patch BER. Additionally, single-molecule studies of FEN1 in complex with PCNA have demonstrated how PCNA boosts FEN1’s activity (57). PCNA serves not only as a processivity factor for Pol δ/ε by forming a clamp around DNA, but also helps maintain the DNA substrate in an open flap conformation, pivotal for FEN1’s function. This enhancement likely involves threading the 5’ single-stranded DNA flap, which improves FEN1’s substrate recognition and increases its catalytic efficiency in the FEN1/PCNA/DNA complex.

UV-DDB

Recent studies have indicated that ultraviolet-damaged DNA-binding protein (UV-DDB), traditionally recognized for its role in nucleotide excision repair (NER), also plays a vital role in BER (58). UV-DDB serves as a general damage sensor by binding to various DNA lesions, including AP sites and 8-oxoguanine (8-oxoG), common types of DNA damage in BER. Single-molecule studies have shown that UV-DDB can enhance the activities of BER enzymes such as OGG1 and APE1. Specifically, UV-DDB increases the cleavage activities of both OGG1 and APE1, substantially improving subsequent gap filling by DNA Pol β by up to 30-fold. This enhancement occurs through transient interactions where UV-DDB assists in the disassociation of glycosylases from DNA, preventing product inhibition and promoting efficient repair turnover. Additionally, UV-DDB has been shown to interact with MUTYH glycosylase, significantly enhancing its efficiency in removing adenines mismatched with 8-oxoG (59). Single-molecule fluorescence studies have indicated that UV-DDB reduces the residence time of MUTYH on AP sites, suggesting that it facilitates MUTYH’s release after the excision process and thus advancing the repair pathway. This highlights UV-DDB’s role in enhancing the base excision repair (BER) process by improving the efficiency of damage recognition and processing. These findings underscore the interconnectedness of DNA repair pathways and suggest that proteins like UV-DDB might have broader roles beyond their originally identified functions. By acting as a universal sensor and enhancer of BER enzyme activities, UV-DDB contributes to the robustness of the DNA repair machinery, ensuring genomic stability amidst various types of DNA damage. Moreover, other proteins that bind strongly to DNA structures generated during BER—yet to be studied extensively—may perform roles akin to UV-DDB. Single-molecule studies could prove crucial in exploring these proteins, providing valuable insights into their functions and potentially unveiling new facets of DNA repair mechanisms.

DISCUSSION

Recent advances in single-molecule techniques have significantly enhanced our understanding of the BER pathway. These techniques facilitate the real-time visualization of individual protein-DNA interactions, providing invaluable insights into the dynamics of DNA glycosylases, AP endonucleases, and other associated proteins in BER. In conclusion, single-molecule studies offer groundbreaking insights into the molecular mechanics of BER, revealing the complex ways in which repair proteins collaborate to maintain genomic stability. Beyond contributing to basic science, these findings hold potential for developing targeted therapies for diseases like cancer, where defects in DNA repair mechanisms are often implicated. Further exploration of these protein-DNA interactions at the single-molecule level is likely to yield additional revelations about the regulation and coordination of the BER pathway, offering novel therapeutic targets for future clinical applications (60-62).

ACKNOWLEDGEMENTS

This research was supported by the KAIST Startup grant (G04230037), Grand Challenge 30 Project, KDDF (RS-2024-00463605) and National Research Foundation of the Korea (NRF-2023R1A2C3006934 and RS-2024-00341654) to G.L..

CONFLICTS OF INTEREST

The authors have no conflicting interests.

FIGURES
Fig. 1. Short-patch and long-patch BER pathways. The figure illustrates the short-and long-patch pathways of BER. In both pathways, a DNA glycosylase initiates the repair by recognizing and removing a damaged base, leaving an AP site. APE1 subsequently cleaves the DNA backbone at the AP site, resulting in a single-strand break. In the short-patch pathway (left), XRCC1 forms a complex with Pol β and LIG3 to replace the damaged nucleotide and seal the nick. In the long-patch pathway (right), PCNA and PARP1 are recruited to the repair site. Pol δ/ε extends the repair patch by displacing the damaged strand. FEN1 then cleaves the displaced flap, and LIG1 seals the remaining nick to complete the repair.
Fig. 2. Single-molecule techniques for studying BER. (A) single-molecule FRET is utilized to monitor conformational changes or interactions between proteins and DNA at the single-molecule level, providing real-time insights into dynamic processes by measuring fluorescence resonance energy transfer between donor and acceptor dyes. (B) Single-molecule DNA tightrope assay involves suspending DNA between two beads or surfaces, forming a “tightrope.” This setup enables the visualization of protein interactions with DNA, allowing observation of their movement and activity along the DNA strand using fluorescence microscopy. (C) Optical tweezers employ focused laser beams to manipulate single-molecules of DNA or proteins by applying picoNewton (pN) forces, making it ideal for examining mechanical properties and forces involved in BER-related processes. (D) Single-molecule flow stretch assay uses tethered DNA molecules on a surface subject to controlled flow, stretching the DNA under the exerted force to facilitate observation of protein-DNA interactions and repair activities.
TABLE

Proteins involved in Base Excision Repair (BER) in E. coli and humans

Type E.coli Human
DNA glycosylase

Ung

Mug

AlkA

TagA

MutY

Nth1

Fpg

Nei

UNG

TDG

SMUG1

MBD4

MPG

MYH

NTH

OGG1

NEIL1

NEIL2

NEIL3

AP endonuclease

ExoIII

Nfo

APE1

APE2

DNA polymerase Pol I

POL β

POL ε

POL δ

DNA Ligase LigA

LIG1

LIG3

Others

FEN1

PCNA

XRCC1

PARP1

UV-DDB

The table lists DNA glycosylases, AP endonucleases, DNA polymerases, DNA ligases, and other related proteins in E. coli and humans.


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