A nucleotide-analogue-induced gain of function corrects the error-prone nature of human DNA polymerase iota.
Y-family DNA polymerases participate in replication stress and DNA damage tolerance mechanisms. The properties that allow these enzymes to copy past bulky adducts or distorted template DNA can result in a greater propensity for them to make mistakes. Of the four human Y-family members, human DNA polymerase iota (hpol ι) is the most error-prone. In the current study, we elucidate the molecular basis for improving the fidelity of hpol ι through use of the fixed-conformation nucleotide North-methanocarba-2'-deoxyadenosine triphosphate (N-MC-dATP). Three crystal structures were solved of hpol ι in complex with DNA containing a template 2'-deoxythymidine (dT) paired with an incoming dNTP or modified nucleotide triphosphate. The ternary complex of hpol ι inserting N-MC-dATP opposite dT reveals that the adenine ring is stabilized in the anti orientation about the pseudo-glycosyl torsion angle, which mimics precisely the mutagenic arrangement of dGTP:dT normally preferred by hpol ι. The stabilized anti conformation occurs without notable contacts from the protein but likely results from constraints imposed by the bicyclo[3.1.0]hexane scaffold of the modified nucleotide. Unmodified dATP and South-MC-dATP each adopt syn glycosyl orientations to form Hoogsteen base pairs with dT. The Hoogsteen orientation exhibits weaker base-stacking interactions and is less catalytically favorable than anti N-MC-dATP. Thus, N-MC-dATP corrects the error-prone nature of hpol ι by preventing the Hoogsteen base-pairing mode normally observed for hpol ι-catalyzed insertion of dATP opposite dT. These results provide a previously unrecognized means of altering the efficiency and the fidelity of a human translesion DNA polymerase.
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205-7199, USA.
Amit
A
Ketkar
Maroof K
MK
Zafar
Surajit
S
Banerjee
Victor E
VE
Marquez
Martin
M
Egli
Robert L
RL
Eoff
D004249:DNA Damage; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D009711:Nucleotides / Q000737:chemistry / Q000235:genetics*; D000094769:DNA Polymerase iota
D009711:Nucleotides; D004259:DNA-Directed DNA Polymerase; D000094769:DNA Polymerase iota; C000722070:POLI protein, human