Pre-steady-state kinetic investigation of bypass of a bulky guanine lesion by human Y-family DNA polymerases.
3-Nitrobenzanthrone (3-NBA), a byproduct of diesel exhaust, is highly present in the environment and poses a significant health risk. Exposure to 3-NBA results in formation of N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dGC8-N-ABA), a bulky DNA lesion that is of particular importance due to its mutagenic and carcinogenic potential. If not repaired or bypassed during genomic replication, dGC8-N-ABAcan stall replication forks, leading to senescence and cell death. Here we used pre-steady-state kinetic methods to determine which of the four human Y-family DNA polymerases (hPolη, hPolκ, hPolι, or hRev1) are able to catalyze translesion synthesis of dGC8-N-ABAin vitro. Our studies demonstrated that hPolη and hPolκ most efficiently bypassed a site-specifically placed dGC8-N-ABAlesion, making them good candidates for catalyzing translesion synthesis (TLS) of this bulky lesion in vivo. Consistently, our publication (Biochemistry 53, 5323-31) in 2014 has shown that small interfering RNA-mediated knockdown of hPolη and hPolκ in HEK293T cells significantly reduces the efficiency of TLS of dGC8-N-ABA. In contrast, hPolι and hRev1 were severely stalled by dGC8-N-ABAand their potential role in vivo was discussed. Subsequently, we determined the kinetic parameters for correct and incorrect nucleotide incorporation catalyzed by hPolη at various positions upstream, opposite, and downstream from dGC8-N-ABA. Notably, nucleotide incorporation efficiency and fidelity both decreased significantly during dGC8-N-ABAbypass and the subsequent extension step, leading to polymerase pausing and error-prone DNA synthesis by hPolη. Furthermore, hPolη displayed nucleotide concentration-dependent biphasic kinetics at the two polymerase pause sites, suggesting that multiple enzyme•DNA complexes likely exist during nucleotide incorporation.
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA; The Ohio State Biophysics Program, The Ohio State University, Columbus, OH 43210, USA. Electronic address: tokarsky.8@osu.edu.
E John
EJ
Tokarsky
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA; The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA. Electronic address: gadkari.3@osu.edu.
Varun V
VV
Gadkari
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA; The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA. Electronic address: zahurancik.2@osu.edu.
Walter J
WJ
Zahurancik
Department of Chemistry, University of Connecticut, Storrs, CT 06269, USA. Electronic address: malikchanchal@gmail.com.
Chanchal K
CK
Malik
Department of Chemistry, University of Connecticut, Storrs, CT 06269, USA. Electronic address: ashis.basu@uconn.edu.
Ashis K
AK
Basu
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA; The Ohio State Biophysics Program, The Ohio State University, Columbus, OH 43210, USA; The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA. Electronic address: suo.3@osu.edu.
Zucai
Z
Suo
D001551:Benz(a)Anthracenes / Q000378:metabolism / Q000494:pharmacology*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018736:DNA Adducts / Q000096:biosynthesis; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D009153:Mutagens / Q000378:metabolism / Q000494:pharmacology*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D000094769:DNA Polymerase iota; D000099270:Y-Family DNA Polymerases
D001551:Benz(a)Anthracenes; D004247:DNA; D018736:DNA Adducts; D004259:DNA-Directed DNA Polymerase; D006147:Guanine; D009153:Mutagens; D009687:Nuclear Proteins; D009713:Nucleotidyltransferases; D000094769:DNA Polymerase iota; C429004:REV1 protein, human; D000099270:Y-Family DNA Polymerases; C404302:POLK protein, human; C112338:Rad30 protein; C117220:3-nitrobenzanthrone; C000722070:POLI protein, human
3-Nitrobenzanthrone; DNA repair; DNA replication; Human DNA polymerase eta; Translesion synthesis