Mechanistic Basis for the Bypass of a Bulky DNA Adduct Catalyzed by a Y-Family DNA Polymerase.
1-Nitropyrene (1-NP), an environmental pollutant, induces DNA damage in vivo and is considered to be carcinogenic. The DNA adducts formed by the 1-NP metabolites stall replicative DNA polymerases but are presumably bypassed by error-prone Y-family DNA polymerases at the expense of replication fidelity and efficiency in vivo. Our running start assays confirmed that a site-specifically placed 8-(deoxyguanosin-N(2)-yl)-1-aminopyrene (dG(1,8)), one of the DNA adducts derived from 1-NP, can be bypassed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), although this representative Y-family enzyme was paused strongly by the lesion. Pre-steady-state kinetic assays were employed to determine the low nucleotide incorporation fidelity and establish a minimal kinetic mechanism for the dG(1,8) bypass by Dpo4. To reveal a structural basis for dCTP incorporation opposite dG(1,8), we solved the crystal structures of the complexes of Dpo4 and DNA containing a templating dG(1,8) lesion in the absence or presence of dCTP. The Dpo4·DNA-dG(1,8) binary structure shows that the aminopyrene moiety of the lesion stacks against the primer/template junction pair, while its dG moiety projected into the cleft between the Finger and Little Finger domains of Dpo4. In the Dpo4·DNA-dG(1,8)·dCTP ternary structure, the aminopyrene moiety of the dG(1,8) lesion, is sandwiched between the nascent and junction base pairs, while its base is present in the major groove. Moreover, dCTP forms a Watson-Crick base pair with dG, two nucleotides upstream from the dG(1,8) site, creating a complex for "-2" frameshift mutation. Mechanistically, these crystal structures provide additional insight into the aforementioned minimal kinetic mechanism.
Rajan
R
Vyas
Georgia
G
Efthimiopoulos
E John
EJ
Tokarsky
Department of Chemistry, University of Connecticut , Storrs, Connecticut 06269, United States.
Chanchal K
CK
Malik
Department of Chemistry, University of Connecticut , Storrs, Connecticut 06269, United States.
Ashis K
AK
Basu
Zucai
Z
Suo
D001483:Base Sequence; D055162:Biocatalysis; D018736:DNA Adducts / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019951:DNA Polymerase beta / Q000378:metabolism*; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011721:Pyrenes / Q000378:metabolism; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology
D018736:DNA Adducts; D003845:Deoxycytosine Nucleotides; D011721:Pyrenes; C024107:2'-deoxycytidine 5'-triphosphate; D019951:DNA Polymerase beta; D008274:Magnesium; C032668:1-nitropyrene