Stalled Replication Fork: Mechanisms, Consequences, and Recovery

Cells must accurately duplicate their DNA to maintain genetic stability, but this process is frequently challenged by obstacles that stall replication forks. If not properly managed, stalled forks can lead to mutations, chromosomal rearrangements, or cell death, contributing to diseases like cancer. Understanding how cells respond to these disruptions is essential for uncovering therapeutic targets and improving genome maintenance strategies.

Causes Of Fork Stalling

Replication forks stall when obstacles impede DNA polymerases, disrupting normal replication and requiring specialized mechanisms to prevent genome instability. Several factors contribute to fork stalling, including DNA lesions, replication stress, and transcription interference.

DNA Damage

DNA damage is a major cause of replication fork stalling, arising from metabolic byproducts or environmental factors like ultraviolet (UV) radiation and chemical mutagens. Lesions such as thymine dimers from UV exposure or bulky adducts from carcinogens like benzo[a]pyrene physically obstruct replication. Oxidative stress generates reactive species that induce strand breaks, further hindering fork progression. Alkylating agents like methyl methanesulfonate (MMS) modify DNA bases, blocking polymerase movement and necessitating repair. Unresolved lesions can cause fork collapse, increasing the risk of chromosomal aberrations and mutagenesis.

Replication Stress

Replication stress challenges DNA duplication, often leading to fork stalling. Nucleotide depletion, caused by insufficient dNTP pools, slows replication. Hydroxyurea, a ribonucleotide reductase inhibitor, induces replication stress by reducing nucleotide availability. Tightly packed chromatin and highly repetitive sequences, such as common fragile sites, also impede fork progression. Oncogene activation can trigger excessive replication initiation, straining replication resources and increasing fork stalling. Persistent replication stress fosters genomic instability, a driver of cancer progression.

Conflicts With Transcription

Replication and transcription occurring in the same genomic regions can hinder fork progression. Encounters with active transcription units create conflicts due to RNA polymerase complexes and R-loops—RNA-DNA hybrids that stabilize stalled transcription machinery. Head-on collisions generate torsional stress, increasing fork arrest risk. Highly transcribed genes, such as ribosomal DNA and certain oncogenes, are hotspots for these conflicts. Cells use helicases like Senataxin and topoisomerases to resolve these obstacles, maintaining replication and transcription coordination. Persistent conflicts can trigger DNA breaks and genomic rearrangements, contributing to neurodegenerative disorders and cancer.

Effects On Genome Integrity

Stalled replication forks pose a serious threat to genome stability by creating single-stranded DNA (ssDNA) regions vulnerable to breakage and mutagenesis. Persistent stalling can activate DNA damage response pathways, leading to fork collapse and double-strand breaks (DSBs), which cause chromosomal translocations and large-scale deletions. These structural changes disrupt gene function, potentially inactivating tumor suppressors or amplifying oncogenes, both key drivers of cancer.

Replication-associated mutagenesis is another consequence. When replication restarts improperly, errors such as nucleotide misincorporation and small insertions or deletions (indels) can occur. Unresolved secondary structures at stalled forks increase the likelihood of template switching, leading to genomic alterations that affect protein function and gene expression.

Replication stress also impacts chromosome segregation during cell division. Under-replicated regions can form ultrafine bridges between segregating chromosomes, increasing the risk of mis-segregation, aneuploidy, and micronucleus formation. Aneuploidy, an abnormal chromosome number, is frequently observed in cancer and linked to poor clinical outcomes. Micronuclei, containing mis-segregated chromosome fragments, amplify DNA damage through aberrant replication and repair mechanisms.

Pathways That Reactivate The Fork

Cells have evolved mechanisms to restart stalled replication forks and prevent genomic instability. Depending on the obstacle, they employ fork reversal, template switching, or translesion synthesis to restore replication.

Fork Reversal

Fork reversal stabilizes stalled replication forks by remodeling them into four-way junctions resembling Holliday junctions. This allows repair enzymes to resolve issues before replication resumes. Proteins like SMARCAL1, ZRANB3, and HLTF facilitate fork remodeling, while nucleases such as MUS81 and RECQ helicases restore normal fork structure once the obstacle is cleared. This strategy prevents fork collapse and double-strand breaks. However, excessive fork remodeling can lead to degradation by nucleases like MRE11, contributing to genomic instability and cancer.

Template Switching

Template switching enables replication to bypass lesions or structural barriers without introducing mutations. This homologous recombination-based mechanism temporarily uses the sister chromatid as a template. RAD51 facilitates strand invasion and template exchange, allowing replication to continue. Template switching is particularly useful for resolving replication stress at repetitive sequences and secondary structures like G-quadruplexes. While generally error-free, improper regulation can cause genomic rearrangements, including duplications and deletions. Defects in this pathway are linked to diseases like Fanconi anemia, characterized by chromosomal instability and increased cancer risk.

Translesion Synthesis

When bulky DNA lesions stall replication forks, translesion synthesis (TLS) enables bypass using specialized DNA polymerases such as Pol η, Pol ι, and Pol κ. These polymerases accommodate distorted DNA structures, allowing replication to continue. However, TLS polymerases have higher error rates than replicative polymerases, increasing the risk of mutations. Cells regulate TLS by ubiquitinating PCNA (proliferating cell nuclear antigen) to recruit TLS polymerases only when necessary. Mutations in TLS polymerases, particularly Pol η, are associated with xeroderma pigmentosum variant (XP-V), a disorder marked by extreme UV sensitivity and increased skin cancer risk due to defective lesion bypass.

Links To Disease States

Failure to manage stalled replication forks has profound implications for human health, particularly in diseases driven by genomic instability. Cancer is a well-documented outcome, as replication stress and improper fork recovery lead to mutations, chromosomal rearrangements, and aneuploidy. Tumor cells often have defects in fork stabilization, with mutations in repair proteins like BRCA1, BRCA2, and FANCD2 impairing fork protection and increasing DNA damage susceptibility. These deficiencies not only drive tumorigenesis but also influence treatment responses. Cancers with defective fork stabilization are more sensitive to DNA-damaging agents like PARP inhibitors, which exploit replication vulnerabilities to induce cell death.

Neurodegenerative disorders are also linked to replication fork dysfunction, particularly diseases involving unstable repeat expansions. Conditions like Huntington’s disease and fragile X syndrome result from trinucleotide repeat expansions, which form secondary structures that stall replication. Improper processing of stalled forks at these sequences can lead to further expansions, worsening disease severity over generations. Inefficient resolution of these replication challenges contributes to neuronal dysfunction and degeneration, demonstrating the broader impact of replication stress beyond proliferative tissues.