cb9b00365_si_002.avi (16.54 MB)
Transcriptional Bypass of DNA–Protein and DNA–Peptide Conjugates by T7 RNA Polymerase
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posted on 2019-10-20, 19:29 authored by Shaofei Ji, Jenna Thomforde, Colette Rogers, Iwen Fu, Suse Broyde, Natalia Y. TretyakovaDNA–protein
cross-links (DPCs) are unusually bulky DNA adducts
that block the access of proteins to DNA and interfere with gene expression,
replication, and repair. We previously described DPC formation at
the N7-guanine position of DNA in human cells treated with antitumor
nitrogen mustards and platinum compounds and have shown that DPCs
can form endogenously at DNA epigenetic mark 5-formyl-dC. However,
insufficient information is available about the effects of these structurally
distinct DPCs on transcription. In the present work, we employ a combination
of in vitro assays, mass spectrometry, and molecular
dynamics simulations to examine the ability of phage T7 RNA polymerase
to bypass DPCs conjugated to the C7 position of 7-deaza-dG and the
C5 position of dC. These model adducts represent endogenous DPCs induced
by exposure to antitumor drugs and formed at epigenetics DNA marks,
respectively. Our results reveal that DPCs containing full-length
proteins significantly inhibit in vitro transcription
by T7 RNA polymerase, while short DNA–peptide cross-links (DpCs)
are bypassed. DpCs conjugated to the C7 position of 7-deaza-dG are
transcribed with high fidelity, while the same polypeptides attached
to the C5 position of dC induce transcription errors. Molecular dynamics
simulations of DpCs conjugated either to the C5 atom of dC or the
C7 position of 7-deaza-dG on the template strand in T7 RNA polymerase
explain how the conjugated peptide can be accommodated in the narrow
major groove of the DNA–RNA hybrid and how the modified dC
can form a stable mismatch with the incoming ATP in the polymerase
active site, allowing for transcriptional mutagenesis.