Single Molecular Resolution to Monitor DNA Replication Fork Dynamics upon Stress by DNA Fiber Assay

Abstract

DNA replication always encounters numerous endogenous and exogenous stresses during the cell cycle. Measuring the cell responses to stress has primarily relied on cell survival and incorporation of radioactive dNTPs, which is limited in resolution. A higher resolution is required to monitor how replication and repair respond to these stresses. This protocol describes a procedure to monitor the length of new synthesized DNA in a single molecular resolution called DNA fiber assay. The fiber assay relies on labeling of nascent DNA with the nucleoside analog 5-Chloro-2'-deoxyuridine (CldU) and 5-Iodo-2'-deoxyuridine (IdU). We can visualize the labeled nascent DNA in single molecular resolution by immunostaining. By measuring labeled DNA length, the assay permits interrogation of replication speed at given conditions, end processing at the reversed fork, and fork restart after repair.

Keywords: DNA fiber assay; Fork degradation; Fork restart; Fork speed; Nascent DNA strand degradation.