DNA Replication Termination: Key Steps and Biological Impact

Cells must accurately duplicate their DNA before division to maintain genetic integrity. While much attention is given to replication initiation and elongation, termination is equally critical. Proper completion prevents genomic instability, ensuring chromosomes are fully replicated without errors or entanglements. Understanding this process provides insight into genome maintenance and its implications for diseases like cancer.

Termination Sites and Sequences

DNA replication concludes at defined termination sites to ensure complete genome duplication. These regions contain specific sequences that signal the replisome to stop. In bacteria like Escherichia coli, ter sites bound by Tus proteins create a directional block preventing over-replication. These sequences are strategically positioned to control fork convergence and avoid genomic instability.

In eukaryotes, termination is influenced more by replication origin positioning and fork progression timing than discrete sequences. High-throughput sequencing has identified preferential termination zones where forks slow and merge, often coinciding with highly transcribed genes, repetitive elements, or chromatin structures that naturally impede movement. G-quadruplexes and R-loops in these areas require specialized helicases and topoisomerases for resolution.

Disruptions in termination efficiency can lead to incomplete replication, fork stalling, or excessive recombination, increasing the risk of chromosomal rearrangements. In yeast, mutations affecting termination zones are linked to replication stress and genome instability. Similarly, defects in termination processes in human cells contribute to cancers where replication stress drives genomic alterations.

Replisome Disassembly Mechanism

Once replication concludes, the replisome must be dismantled to prevent interference with cellular processes. This involves removing replication-associated proteins to avoid persistent intermediates that could trigger DNA damage responses or hinder chromosome segregation.

A key player in replisome disassembly is the ATPase p97 (VCP/CDC48), which, along with cofactors UFD1 and NPL4, recognizes ubiquitylated replisome components and extracts them from chromatin. Ubiquitylation of the CMG helicase complex (Cdc45, MCM2-7, and GINS) signals p97 for helicase disassembly. In Saccharomyces cerevisiae, the E3 ubiquitin ligase SCFDia2 tags CMG for removal, ensuring helicase clearance.

In prokaryotes, replisome disassembly follows a distinct but regulated pathway. The DnaB helicase must be released once replication ends, facilitated by proteins like Hda, which interacts with the DNA polymerase clamp (β-clamp) to promote disassembly. Hda stimulates ATP hydrolysis by the clamp loader DnaA, inactivating the replication machinery. RecG helicase further resolves lingering replication intermediates, ensuring a smooth transition to other chromosomal processes.

Role of Helicases in Stalling and Release

Helicases manage stalled forks and ensure their resolution. These enzymes unwind DNA ahead of the replication machinery but must also address obstacles that impede fork progression. Without proper regulation, stalled forks may collapse, leading to incomplete replication and genomic instability.

Pif1 helicase resolves G-quadruplexes—four-stranded DNA structures that form in guanine-rich regions and hinder replication. In Saccharomyces cerevisiae, Pif1 deficiency increases fork stalling at these motifs, causing replication stress. Human homologs like PIF1 and BLM perform similar roles, reducing the risk of stalled replication. BLM, a RecQ helicase, is particularly important in resolving complex DNA intermediates and preventing fork collapse.

Helicases also coordinate with checkpoint signaling pathways to manage stalled forks. ATR kinase, a key regulator of replication stress, interacts with helicases like WRN to stabilize arrested forks and facilitate restart. WRN mutations are linked to Werner syndrome, a disorder characterized by premature aging and genomic instability. In bacteria, helicases like UvrD and Rep remove protein obstacles from DNA, preventing prolonged replication arrest. UvrD helps dislodge RNA polymerase when transcription-replication conflicts arise, ensuring smooth fork progression.

Fork Merging at Convergence Zones

As replication progresses, forks advance until they meet at convergence zones. These merging events must be carefully coordinated to prevent errors like over-replication or incomplete synthesis. Fork speed and stability are influenced by chromatin structure, replication timing, and DNA-bound proteins. Single-molecule tracking studies show that fork velocity adjusts in response to obstacles, ensuring synchronized convergence.

When forks meet, DNA polymerase extends synthesis until the leading strands merge, followed by ligases sealing the final nicks. This process is particularly critical in repetitive sequences, where misalignment could cause small deletions or duplications. Research in Drosophila links disruptions in fork merging to fragile site instability and increased chromosomal breakage. Specialized nucleases like Mus81-Eme1 resolve lingering replication intermediates, preventing interference with chromosome segregation.

Chromosome Decatenation

After forks merge, newly synthesized sister chromatids often remain intertwined, requiring decatenation before segregation. Type II topoisomerases introduce transient double-strand breaks to untangle DNA. In eukaryotes, topoisomerase IIα plays a key role, passing one duplex through another before resealing breaks. Its activity peaks in late S and G2 phases to ensure complete resolution before mitosis.

In prokaryotes, DNA gyrase and topoisomerase IV handle decatenation, with topoisomerase IV playing a primary role in unlinking replicated circular chromosomes. Their coordinated action prevents chromosome dimers, which can obstruct segregation. In Escherichia coli, topoisomerase IV impairments lead to highly intertwined DNA and cell division defects. Bacterial topoisomerase inhibitors like fluoroquinolones exploit this mechanism by trapping the enzyme-DNA complex, inducing lethal double-strand breaks.

Consequences of Misregulation

Failures in replication termination lead to genomic instability, replication stress, and increased mutation susceptibility. Inefficient termination can cause persistent forks to collapse, triggering double-strand breaks requiring error-prone repair pathways. This contributes to chromosomal rearrangements, deletions, or duplications frequently observed in cancer genomes. Defects in termination factors like TRAIP, a ubiquitin ligase involved in replisome disassembly, are linked to increased replication stress and mitotic abnormalities in human cells.

Beyond cancer, termination defects are implicated in genetic disorders associated with genome fragility. Mutations in helicases like BLM, linked to Bloom syndrome, result in excessive recombination and sister chromatid exchanges due to unresolved replication intermediates. Similarly, topoisomerase deficiencies are associated with neurodevelopmental disorders, highlighting the importance of proper termination in maintaining genomic integrity in rapidly dividing cells.