The DNA fibre technique - tracking helicases at work.
Faithful duplication of genetic material during every cell division is essential to ensure accurate transmission of genetic information to daughter cells. DNA helicases play a crucial role in promoting this process by facilitating almost all transactions occurring on DNA, including DNA replication and repair. They are responsible not only for DNA double helix unwinding ahead of progressing replication forks but also for resolution of secondary structures like G4 quadruplexes, HJ branch migration, double HJ dissolution, protein displacement, strand annealing and many more. Their importance in maintaining genome stability is underscored by the fact that many human disorders, including cancer, are associated with mutations in helicase genes. Here we outline how DNA fibre fluorography, a straightforward and inexpensive approach, can be employed to study the in vivo function of helicases in DNA replication and the maintenance of genome stability at a single molecule level. This approach directly visualizes the progression of individual replication forks within living cells and hence provides quantitative information on various aspects of DNA synthesis, such as replication fork processivity (replication speed), fork stalling, origin usage and fork termination.
Department of Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK. Electronic address: jadwiga.nieminuszczy@oncology.ox.ac.uk.
Jadwiga
J
Nieminuszczy
Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.
Rebekka A
RA
Schwab
Department of Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.
Wojciech
W
Niedzwiedz
D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics; D004261:DNA Replication / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics; D054856:G-Quadruplexes; D005818:Genetic Engineering / Q000379:methods*; D042822:Genomic Instability / Q000235:genetics; D006801:Humans
D004277:DNA, Single-Stranded; D004265:DNA Helicases
DNA fibre analysis; DNA replication; Fork stalling; Helicase; Origin firing; Replication speed