Publication Date
4-3-2023
Journal
Journal of Cell Biology
DOI
10.1083/jcb.202207121
PMID
36795402
PMCID
PMC9960042
PubMedCentral® Posted Date
2-16-2023
PubMedCentral® Full Text Version
Post-print
Published Open-Access
yes
Keywords
Humans, DNA, DNA Replication, Escherichia coli, Tandem Mass Spectrometry, Thymidine, Cell Nucleus, Mitochondria
Abstract
The primary method for probing DNA replication dynamics is DNA fiber analysis, which utilizes thymidine analog incorporation into nascent DNA, followed by immunofluorescent microscopy of DNA fibers. Besides being time-consuming and prone to experimenter bias, it is not suitable for studying DNA replication dynamics in mitochondria or bacteria, nor is it adaptable for higher-throughput analysis. Here, we present mass spectrometry-based analysis of nascent DNA (MS-BAND) as a rapid, unbiased, quantitative alternative to DNA fiber analysis. In this method, incorporation of thymidine analogs is quantified from DNA using triple quadrupole tandem mass spectrometry. MS-BAND accurately detects DNA replication alterations in both the nucleus and mitochondria of human cells, as well as bacteria. The high-throughput capability of MS-BAND captured replication alterations in an E. coli DNA damage-inducing gene library. Therefore, MS-BAND may serve as an alternative to the DNA fiber technique, with potential for high-throughput analysis of replication dynamics in diverse model systems.
Included in
Biological Phenomena, Cell Phenomena, and Immunity Commons, Biomedical Informatics Commons, Genetics and Genomics Commons, Medical Genetics Commons, Medical Molecular Biology Commons, Medical Specialties Commons