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Assessing SMC Complex Function in Replication Fork Progression with DNA Fiber Assays.

Neus P Lorite1,2, Snehlata1, Neus Colomina1

  • 1Departament de Ciències Mèdiques Bàsiques, Institut de Recerca Biomèdica de Lleida, Universitat de Lleida, Lleida, Spain.

Methods in Molecular Biology (Clifton, N.J.)
|October 19, 2025
PubMed
Summary
This summary is machine-generated.

This study details a DNA fiber assay protocol to analyze DNA replication dynamics and fork progression in human cells. The method is applied to investigate Structural Maintenance of Chromosomes (SMC) complex roles in replication, particularly under stress.

Keywords:
ChromosomeCldUDNA fiberDNA repairDNA replicationHydroxyureaIdUNSE1NSMCE1Replication forkReplicative stressSMC5/6

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genetics

Background:

  • Chromosome organization changes significantly during S phase as DNA replicates.
  • Structural Maintenance of Chromosomes (SMC) complexes are crucial for organizing chromatin during replication and responding to stress.
  • Understanding replication dynamics is key to comprehending cell division and genomic stability.

Purpose of the Study:

  • To present a detailed protocol for the DNA fiber assay in human cell lines.
  • To provide a quantitative method for analyzing replication fork progression and dynamics.
  • To investigate the role of SMC complexes in regulating DNA replication, especially under replicative stress.

Main Methods:

  • Sequential incorporation of two nucleotide analogs into cellular DNA.
  • DNA spreading and immunodetection to visualize replicated DNA tracks.
  • Quantitative analysis of DNA fiber lengths to determine replication fork rates.

Main Results:

  • The DNA fiber assay protocol was successfully established for human cell lines.
  • The assay allowed for quantitative analysis of replication fork progression in wild-type and SMC5/6 mutant cells.
  • Replicative stress induced by hydroxyurea was effectively studied using this method.

Conclusions:

  • The DNA fiber assay is a versatile and quantitative technique for studying DNA replication dynamics.
  • This method facilitates the investigation of SMC complex functions in replication fork progression and stress response.
  • The protocol can be adapted to study various aspects of replication, including fork reversal, restart, and protection.