1. UBA1 is recruited to DNA and promotes Chk1 phosphorylation.
UBA1 is recruited to DNA and promotes Chk1 phosphorylation. (A) Nuclear extracts prepared from HEK293 cells expressing UBA1-FLAG were incubated with the indicated biotinylated DNA substrates. DNA was pulled down using streptavidin-coated beads, resolved by SDS–PAGE, and probed for FLAG-UBA1 by Western blotting using anti-FLAG and anti-UBA1 antibodies, as indicated. (B) Nuclear extracts were incubated with recombinant FLAG-ubiquitin and the indicated biotinylated DNA substrates. DNA-bound ubiquitin conjugates were pulled down using streptavidin-coated beads and revealed by Western blotting using anti-FLAG antibody. (C) HeLa S3 cells were exposed to methyl methanesulfonate (MMS, 10 μg/ml) for 60 min in the presence of increasing concentration of PYR41 and probed for Chk1 and phospho-Chk1 (Ser345) by Western blotting. (D) Nuclear extracts were incubated with gapped DNA in the presence of [32P]-labelled NAD. Poly(ADP-ribosyl)ated proteins were resolved by PAGE and revealed by autoradiography. (E) Analysis of UBA1 and PCNA on nascent DNA. When indicated, HEK293 cells were pretreated with PARP1 inhibitors (10 μM of PJ34 or 10 μM of olaparib) and then pulse-labelled with EdU for 10 min. In no click samples (no Clk), desthiobiotin-TEG azide was replaced by DMSO. The fraction of UBA1 and PCNA associated with nascent DNA was retrieved using the iPOND procedure and revealed by Western blotting.
Ramhari Kumbhar et al. LSA 2018;1:e
© 2018 Kumbhar et al.