Bypass of DNA-Protein Cross-links Conjugated to the 7-Deazaguanine Position of DNA by Translesion Synthesis Polymerases.
DNA-protein cross-links (DPCs) are bulky DNA lesions that form both endogenously and following exposure to bis-electrophiles such as common antitumor agents. The structural and biological consequences of DPCs have not been fully elucidated due to the complexity of these adducts. The most common site of DPC formation in DNA following treatment with bis-electrophiles such as nitrogen mustards and cisplatin is the N7 position of guanine, but the resulting conjugates are hydrolytically labile and thus are not suitable for structural and biological studies. In this report, hydrolytically stable structural mimics of N7-guanine-conjugated DPCs were generated by reductive amination reactions between the Lys and Arg side chains of proteins/peptides and aldehyde groups linked to 7-deazaguanine residues in DNA. These model DPCs were subjected to in vitro replication in the presence of human translesion synthesis DNA polymerases. DPCs containing full-length proteins (11-28 kDa) or a 23-mer peptide blocked human polymerases η and κ. DPC conjugates to a 10-mer peptide were bypassed with nucleotide insertion efficiency 50-100-fold lower than for native G. Both human polymerase (hPol) κ and hPol η inserted the correct base (C) opposite the 10-mer peptide cross-link, although small amounts of T were added by hPol η. Molecular dynamics simulation of an hPol κ ternary complex containing a template-primer DNA with dCTP opposite the 10-mer peptide DPC revealed that this bulky lesion can be accommodated in the polymerase active site by aligning with the major groove of the adducted DNA within the ternary complex of polymerase and dCTP.
From the Masonic Cancer Center and.|the Departments of Chemistry.
Susith
S
Wickramaratne
From the Masonic Cancer Center and.|the Departments of Chemistry.
Shaofei
S
Ji
Chemistry, Stony Brook University, Stony Brook, New York 11794.
Shivam
S
Mukherjee
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Yan
Y
Su
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Matthew G
MG
Pence
Department of Biology, New York University, New York, New York 10003-6688, and.
Lee
L
Lior-Hoffmann
Department of Biology, New York University, New York, New York 10003-6688, and.
Iwen
I
Fu
Department of Biology, New York University, New York, New York 10003-6688, and.
Suse
S
Broyde
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
F Peter
FP
Guengerich
the Departments of Chemistry.
Mark
M
Distefano
Chemistry, Stony Brook University, Stony Brook, New York 11794.|Departments of Pharmacological Sciences and.
Orlando D
OD
Schärer
Center for Drug Design and.
Yuk Yin
YY
Sham
From the Masonic Cancer Center and trety001@umn.edu.|Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455.
Natalia
N
Tretyakova
D000586:Amination; D000595:Amino Acid Sequence; D001483:Base Sequence; D018736:DNA Adducts / Q000737:chemistry* / Q000235:genetics; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry; D006801:Humans; D056004:Molecular Dynamics Simulation; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000378:metabolism
D018736:DNA Adducts; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; D006147:Guanine; D004259:DNA-Directed DNA Polymerase; C066856:7-deazaguanine
DNA damage; DNA polymerase; DNA-protein conjugates; gel electrophoresis; mass spectrometry (MS); molecular dynamics; reductive amination; steady-state kinetics; translesion synthesis