Dataset Information

Analysis of Rev1-dependent replication dynamics using isolation of proteins on nascent DNA

ABSTRACT: To study the influence of the Rev1 translesion synthesis enzyme on fork dynamics, we performed isolation of proteins on nascent DNA coupled with mass spectrometry (iPOND-MS). Wild-type and REV1-KO HEK293 Flp-In™/T-REx™ cells were used. Treatment with the G-quadruplex stabilizing agent pyridostatin (PDS; 10 micromolar for either 15 or 60 minutes) was used to examine the Rev1-dependent response to G4-induced replication stress. A thymidine pulse experiment was used to ensure that replication proteins were enriched in the samples pulsed with 5-ethynyl-2ʹ-deoxyuridine (EdU). The resulting samples were analyzed using liquid chromatography mass spectrometry (LC-MS). Label-free quantitative analysis of peptide abundance was used to determine changes in response to Rev1 deletion and treatment with PDS.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Dennis Province