Recombinant Flap endonuclease 1 (FEN1), partial

What is the primary function of FEN1 in DNA metabolism?

FEN1 is a structure-specific endonuclease that evolved to cut at the base of single-stranded flaps in DNA, making it a central component of cellular DNA metabolism. Its substrate specificity allows it to process intermediates in multiple essential DNA pathways .

The primary functions of FEN1 include:

• Processing Okazaki fragments during DNA replication by removing RNA primers

• Participating in long-patch base excision repair (BER)

• Contributing to telomere maintenance

• Assisting in stalled replication fork rescue

FEN1 operates by binding to the flap base, threading the 5′ end of the flap through its helical arch and active site, and then cleaving the flap to create a nick that can be sealed by DNA ligase I . This threading mechanism ensures specificity and prevents indiscriminate nuclease activity on single-stranded regions between Okazaki fragments.

How does FEN1 contribute to Okazaki fragment maturation?

FEN1 plays a crucial role in the accurate maturation of Okazaki fragments during lagging strand DNA synthesis through the following steps:

• DNA polymerase δ (Pol δ) elongates the initiator RNA-DNA primer created by DNA polymerase α/primase

• When Pol δ encounters the downstream Okazaki fragment, it begins strand-displacement synthesis

• This displaces the 5′ end of the preceding Okazaki fragment, creating a single-stranded 5′ flap structure

• FEN1 recognizes this structure, binds to the base of the flap, and precisely cleaves it

• This cleavage removes the RNA primer and sometimes a portion of the initiator DNA, creating a nick

• DNA ligase I then seals the nick, completing the maturation process

This process is often referred to as the "short flap pathway" for Okazaki fragment maturation. FEN1's precision in this process is essential for maintaining genome fidelity, as errors could lead to frame shifts or other mutations.

What experimental models are suitable for studying FEN1 function?

For studying FEN1 function, researchers have employed several experimental models:

• In vitro biochemical assays: Using recombinant FEN1 protein with synthetic DNA substrates containing flap structures. These assays can reveal the enzymatic properties, substrate specificity, and kinetics of FEN1 .

• Cell culture models: Human cell lines (U2OS, HEK293) with FEN1 knockdown or knockout. RNA interference screening has been particularly useful for identifying FEN1's role in DNA repair pathways .

• Yeast models: Saccharomyces cerevisiae has been instrumental in discovering synthetic lethal relationships with FEN1/RAD27 (the yeast homolog), providing insight into potential therapeutic applications .

• Mouse models: Xenograft models established from cancer cell lines have been used to test FEN1 inhibitors in vivo and validate findings from cell culture experiments .

Notably, the development of specialized techniques such as the RADAR (rapid approach to DNA adduct recovery) assay has facilitated the study of FEN1's role in processing DNA-protein crosslinks .

How does the "threading versus tracking" debate influence our understanding of FEN1 mechanism?

The mechanism by which FEN1 processes flap substrates has been subject to debate, with two competing models: the tracking model and the threading model.

The tracking model initially proposed that FEN1 must enter at the free 5′ end of the flap and move along it to reach the base for cleavage. This model was supported by observations that FEN1 activity was inhibited when the 5′ flap contained secondary structures, conjugated streptavidin, or certain chemical adducts .

• FEN1 first recognizes and binds to the flap base junction (the double-strand/single-strand interface)

• The enzyme then threads the 5′ end and subsequently the entire flap through its active site for cleavage

• This threading process is essential for proper positioning of the substrate for catalysis

This mechanistic understanding has important implications:

• It explains FEN1's interaction with PCNA, which strongly stimulates its nuclease activity

• It supports the observation that FEN1 functions as part of larger protein complexes at the flap base

• It reconciles how FEN1 can effectively process substrates without wandering to find the flap 5′ end

The threading mechanism also has implications for how FEN1 might process DNA-protein crosslinks, as large proteins would block threading unless they are first proteolyzed or denatured .

What are the molecular determinants of FEN1's substrate specificity?

FEN1's substrate specificity is determined by several structural features that allow it to recognize and process specific DNA conformations:

• Recognition of the flap base: FEN1 initially recognizes the junction where single-stranded DNA meets double-stranded DNA, binding to the flap base rather than the flap end .

• Helical arch structure: FEN1 contains a helical arch through which the single-stranded flap must thread. This architecture ensures that only proper flap structures can reach the active site .

• Active site configuration: Once threading is complete, the active site positions the substrate for precise cleavage exactly one nucleotide into the double-stranded region.

• Requirement for a free 5′ end: Despite initial binding at the base, FEN1 requires a free 5′ end of the flap to complete threading and cleavage. Experiments show that blocking the 5′ end with bulky adducts inhibits cleavage .

• Accommodation of modified substrates: FEN1 can process substrates with small chemical modifications on the flap, such as amino acid mimetics, but its activity is significantly reduced when processing substrates with bulky proteins attached, such as a streptavidin tetramer .

Understanding these determinants has practical implications for developing FEN1 inhibitors and for engineering FEN1 variants with altered substrate preferences for research purposes.

What is the role of post-translational modifications in regulating FEN1 activity?

Post-translational modifications (PTMs) critically regulate FEN1 activity, with PARylation (poly-ADP-ribosylation) emerging as particularly important:

• PARylation of FEN1:

  • The E285 residue has been identified as a dominant PARylation site in FEN1

  • This modification is crucial for relocating FEN1 to DNA-protein crosslink (DPC) sites

  • PARylation of FEN1 drives its recruitment to damaged DNA sites

• PARylation of FEN1 substrates:

  • PARylation of DPC substrates signals FEN1 recruitment

  • This creates a bidirectional signaling mechanism where both the substrate and FEN1 can be modified to facilitate repair

• Cell cycle-dependent regulation:

  • FEN1 activity appears to be regulated in a cell cycle-dependent manner

  • FEN1 repairs FA-induced DPCs primarily in a replication-dependent manner, with more activity observed in S phase than in G1 phase

• Protein-protein interactions:

  • FEN1 activity is strongly stimulated by interaction with PCNA

  • These interactions are likely regulated by PTMs that modulate binding affinity

The discovery of the PARP1-FEN1 nuclease pathway represents a novel mechanism for repairing diverse DPCs and preventing DPC-induced genomic instability, highlighting how PTMs coordinate complex DNA repair processes .

How does FEN1 contribute to DNA-protein crosslink (DPC) repair?

Recent research has revealed an unexpected role for FEN1 in repairing DNA-protein crosslinks (DPCs), particularly those induced by formaldehyde (FA):

• Alternative to proteolysis pathways: FEN1 provides a nucleolytic mechanism for DPC resolution that operates in parallel to the SPRTN protease pathway. While SPRTN degrades the protein component of the DPC, FEN1 excises the DNA strand containing the crosslink .

• Processing of flaps containing DPCs: FEN1 excises DPCs that are incorporated into 5′-flap structures. These flaps can arise from:

  • The base excision repair (BER) pathway processing oxidative lesions

  • Okazaki fragment maturation during DNA replication

• Requirement for proteolysis: Although FEN1 provides an alternative to the SPRTN pathway, effective DPC processing by FEN1 still requires prior action by the proteasome or p97/VCP to proteolytically process or denature the bulky protein components of the DPC. This facilitates the threading of the flap through FEN1's helical arch .

• Replication-dependent activity: FEN1's DPC repair function appears to be primarily S-phase specific, with significantly more activity observed during DNA replication than in G1 phase .

• Prevention of genomic instability: FEN1 knockdown or knockout cells show increased sensitivity to FA treatment and accumulate more endogenous and spontaneous DPCs, indicating that FEN1 plays a crucial role in preventing DPC-induced genomic instability .

This discovery of FEN1's role in DPC repair represents a significant advancement in understanding how cells cope with these cytotoxic lesions that can block essential DNA transactions.

What is the interplay between FEN1 and PARP1 in DNA repair pathways?

The interplay between FEN1 and PARP1 (poly(ADP-ribose) polymerase 1) represents a sophisticated signaling mechanism for DNA repair:

• PARP1-FEN1 nuclease pathway: Recent research has uncovered a PARP1-FEN1 nuclease pathway that serves as a universal mechanism for repairing diverse DPCs and preventing genomic instability .

• Bidirectional PARylation signaling:

  • PARP1 mediates PARylation of DPC substrates, which serves as a signal for FEN1 recruitment

  • PARP1 also catalyzes PARylation of FEN1 itself, particularly at residue E285

  • This PARylation of FEN1 drives its relocation to DPC sites

• Coordination in oxidative damage repair:

  • FA induces base damage that is processed by the BER pathway

  • This creates 5′-flap structures that contain DPCs

  • The damaged DNA bases colocalize with DPCs and FEN1

  • This suggests a coordinated response where oxidative damage triggers both PARP1 activation and FEN1 recruitment

• Replication stress response:

  • Both PARP1 and FEN1 respond to replication stress

  • PARP1 is among the most abundant proteins found in FA-induced DPCs

  • This suggests that PARP1 may become trapped at sites of replication stress, requiring FEN1 for resolution

This PARP1-FEN1 axis represents an important mechanism for maintaining genome stability, particularly during DNA replication when cells are most vulnerable to DPC formation.

How are oxidative lesions converted to FEN1 substrates in the context of DPC repair?

The conversion of oxidative lesions to FEN1 substrates in the context of DPC repair involves a sophisticated process:

• Base damage induction: Formaldehyde (FA) not only creates DPCs but also damages DNA bases, inducing oxidative lesions .

• BER pathway initiation: These damaged bases are recognized by DNA glycosylases, which remove the damaged base, creating an abasic (AP) site .

• AP site processing: AP endonuclease 1 (APE1) cleaves the phosphodiester backbone 5′ to the AP site, generating a 3′-OH group and a 5′-deoxyribose phosphate (5′-dRP) residue .

• Displacement synthesis: DNA polymerase β (Pol β) or other polymerases perform strand-displacement synthesis, creating a 5′-flap structure that contains the original damage and potentially a crosslinked protein .

• Generation of DPC-containing flaps: This process converts a no-break DPC into a 5′-flap structure containing the DPC, making it a substrate for FEN1 .

Importantly, for FEN1 to effectively process these DPC-containing flaps:

• The proteasome or p97/VCP must first proteolyze or denature the protein component of the DPC

• This reduction in bulk allows the flap to thread through FEN1's helical arch

• FEN1 then cleaves at the base of the flap, removing both the damaged DNA and the crosslinked protein remnant

This mechanism explains how FEN1 can repair DPCs that occur in the middle of DNA molecules, not just those at the 5′ ends of Okazaki fragments.

How can FEN1 be exploited as a therapeutic target for cancer treatment?

FEN1 has emerged as a promising therapeutic target for cancer treatment, particularly for cancers with defects in homologous recombination (HR):

These findings support further investigation of FEN1 inhibitors as potential therapeutic agents, particularly for cancers with HR deficiencies that are often difficult to treat.

What methodological approaches are used to study FEN1 in DNA repair pathways?

Researchers employ various methodological approaches to study FEN1's role in DNA repair pathways:

• RADAR (Rapid Approach to DNA Adduct Recovery) assay:

  • Used to isolate and quantify DNA-protein crosslinks (DPCs)

  • Allows examination of how FEN1 processes these structures

• Biochemical cleavage assays:

  • Utilizing synthetic oligonucleotide substrates with defined flap structures

  • Can be modified with amino acid mimetics or biotin-streptavidin to model DPCs

  • Assessing cleavage products through denaturing PAGE and fluorescent or radioactive labeling

• RNA interference screening:

  • siRNA libraries targeting DNA repair genes

  • Identification of synthetic lethal interactions with DNA-damaging agents

  • Used to identify FEN1 as a critical factor for DPC repair

• Genetic knockout and rescue experiments:

  • CRISPR-Cas9-mediated FEN1 knockout cell lines

  • Rescue experiments with wild-type or mutant FEN1 expression

  • Assessment of cellular sensitivity to DNA-damaging agents

• Fluorescence microscopy techniques:

  • Monitoring subnuclear distribution of proteins using HaloTag expression systems

  • Tracking colocalization of FEN1 with DNA damage sites

  • Analysis of cell cycle-dependent responses to DNA damage

• Immunoprecipitation and mass spectrometry:

  • Identification of FEN1 post-translational modifications

  • Mapping of the E285 PARylation site on FEN1

  • Characterization of FEN1-interacting proteins

These diverse methodological approaches have been instrumental in uncovering FEN1's multifaceted roles in DNA repair and maintenance of genome stability.

What are the most effective ways to inhibit FEN1 activity in experimental settings?

Several approaches have been developed to inhibit FEN1 activity in experimental settings:

• Small molecule inhibitors:

  • FEN1-IN-4 (FEN1i) has been shown to effectively block FEN1 endonuclease activity

  • Small-molecule FEN1 inhibitors have demonstrated selective toxicity against BRCA1/2-deficient cancer cell lines both in vitro and in mouse xenograft models

  • These inhibitors work by targeting FEN1's active site, preventing its flap endonuclease activity

• RNA interference:

  • siRNA knockdown of FEN1 expression has been widely used in research settings

  • This approach has proven effective in sensitizing cells to formaldehyde and in identifying synthetic lethal interactions

  • Multiple independent siRNAs targeting different regions of FEN1 mRNA can be used to ensure specificity

• CRISPR-Cas9 gene editing:

  • Generation of FEN1 knockout cell lines provides a complete loss-of-function model

  • This approach allows for clean genetic experiments and rescue studies with mutant FEN1 variants

  • FEN1 knockout cells accumulate endogenous and spontaneous DPCs even without exogenous damaging agents

• Dominant-negative mutants:

  • Expression of catalytically inactive FEN1 mutants can interfere with endogenous FEN1 function

  • Particularly useful for studying the role of specific FEN1 domains or activities

• Blocking essential post-translational modifications:

  • Targeting the E285 residue of FEN1 that undergoes PARylation can prevent FEN1 recruitment to DNA damage sites

  • This approach allows for specific inhibition of FEN1's role in DPC repair without affecting its other functions

The choice of inhibition method depends on the specific research question, with small molecule inhibitors offering advantages for potential therapeutic applications, while genetic approaches provide cleaner mechanistic insights.

What are the major unresolved questions about FEN1 function in DNA repair?

Despite significant advances in understanding FEN1's roles in DNA metabolism and repair, several important questions remain unresolved:

• Substrate selection mechanism: How does FEN1 distinguish between different types of flap structures in vivo, and what determines its prioritization of certain repair pathways over others?

• Coordination with other repair pathways: The full extent of crosstalk between FEN1-mediated repair and other DNA repair pathways, such as homologous recombination and nucleotide excision repair, requires further clarification .

• Regulation during cell cycle: The precise mechanisms regulating FEN1 activity throughout the cell cycle, particularly during S phase when both replication and repair occur simultaneously, need further investigation .

• Role in specific disease contexts: Beyond BRCA-deficient cancers, the potential role of FEN1 dysregulation in other diseases characterized by genomic instability warrants exploration .

• Structural determinants of DPC processing: The structural features that allow FEN1 to process flaps containing protein adducts, particularly after proteolytic processing, remain incompletely understood .

Addressing these questions will provide deeper insights into FEN1's fundamental biology and potentially reveal new therapeutic applications for FEN1 modulators.

How might advances in FEN1 research impact clinical practice?

Advances in FEN1 research have significant potential to impact clinical practice in several areas:

• Cancer therapeutics:

  • Development of FEN1 inhibitors as targeted therapies for BRCA1/2-deficient cancers

  • Potential combination therapies with existing DNA-damaging agents or PARP inhibitors

  • Biomarker development to identify patients likely to respond to FEN1-targeted therapies

• Diagnostic tools:

  • FEN1 expression or activity levels as biomarkers for cancer prognosis

  • Screening for synthetic lethal interactions with FEN1 to identify potential treatment vulnerabilities

• Understanding disease mechanisms:

  • Insights into how DPC accumulation contributes to aging and neurodegenerative diseases

  • Elucidation of FEN1's role in preventing genomic instability that underlies various pathologies

• Drug resistance mechanisms:

  • Understanding how cancers might develop resistance to FEN1 inhibitors

  • Developing strategies to overcome such resistance

• Personalized medicine approaches:

  • Tailoring treatments based on a patient's FEN1 status and related DNA repair pathway activities

  • Combination therapies targeting multiple DNA repair vulnerabilities