TY - JOUR
T1 - Rapid profiling of DNA replication dynamics using mass spectrometry–based analysis of nascent DNA
AU - Ashour, Mohamed E.
AU - Byrum, Andrea K.
AU - Meroni, Alice
AU - Xia, Jun
AU - Singh, Saurabh
AU - Galletto, Roberto
AU - Rosenberg, Susan M.
AU - Vindigni, Alessandro
AU - Mosammaparast, Nima
N1 - Funding Information:
This work was supported by a fellowship from the American Italian Cancer Foundation (to A. Meroni), the Alvin J. Siteman Cancer Center Siteman Investment Program (supported by The Foundation for Barnes-Jewish Hospital, Cancer Frontier Fund, to A. Vindigni and N. Mosammaparast), the U.S. Department of Defense Breast Cancer Research Program Expansion Award (BC191374 to A. Vindigni), the State of Nebraska (LB595 and LB692 to J. Xia) the National Institutes of Health (K99 ES033259 to J. Xia; R01 CA250905 and DP1 AG072751 to S.M. Rosenberg; R35 GM139508 to R. Galletto; R01 CA237263 and R01 CA248526 to A. Vindigni; R01 CA193318 and P01 CA092584 to N. Mo-sammaparast), an American Cancer Society Research Scholar Award (RSG-18-156-01-DMC to N. Mosammaparast), the Cen-tene Corporation (to N. Mosammaparast), and the Barnard Foundation (to A. Vindigni and N. Mosammaparast).
Publisher Copyright:
© 2023 Ashour et al.
PY - 2023/4/3
Y1 - 2023/4/3
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85148263724&partnerID=8YFLogxK
U2 - 10.1083/jcb.202207121
DO - 10.1083/jcb.202207121
M3 - Article
C2 - 36795402
AN - SCOPUS:85148263724
SN - 0021-9525
VL - 222
JO - Journal of Cell Biology
JF - Journal of Cell Biology
IS - 4
M1 - e202207121
ER -