Structure of human DNA polymerase kappa inserting dATP opposite an 8-OxoG DNA lesion.
BACKGROUND
Oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA and 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major lesions formed. It is amongst the most mutagenic lesions in cells because of its dual coding potential, wherein 8-oxoG(syn) can pair with an A in addition to normal base pairing of 8-oxoG(anti) with a C. Human DNA polymerase kappa (Polkappa) is a member of the newly discovered Y-family of DNA polymerases that possess the ability to replicate through DNA lesions. To understand the basis of Polkappa's preference for insertion of an A opposite 8-oxoG lesion, we have solved the structure of Polkappa in ternary complex with a template-primer presenting 8-oxoG in the active site and with dATP as the incoming nucleotide.


RESULTS
We show that the Polkappa active site is well-adapted to accommodate 8-oxoG in the syn conformation. That is, the polymerase and the bound template-primer are almost identical in their conformations to that in the ternary complex with undamaged DNA. There is no steric hindrance to accommodating 8-oxoG in the syn conformation for Hoogsteen base-paring with incoming dATP.


CONCLUSIONS
The structure we present here is the first for a eukaryotic translesion synthesis (TLS) DNA polymerase with an 8-oxoG:A base pair in the active site. The structure shows why Polkappa is more efficient at inserting an A opposite the 8-oxoG lesion than a C. The structure also provides a basis for why Polkappa is more efficient at inserting an A opposite the lesion than other Y-family DNA polymerases.
Vasquez-Del Carpio
Rodrigo
R
Department of Structural & Chemical Biology, Mount Sinai School of Medicine, New York, NY, USA.
Silverstein
Timothy D
TD
Lone
Samer
S
Swan
Michael K
MK
Choudhury
Jayati R
JR
Johnson
Robert E
RE
Prakash
Satya
S
Prakash
Louise
L
Aggarwal
Aneel K
AK
D000255:Adenosine Triphosphate / Q000737:chemistry*; D020134:Catalytic Domain; D003460:Crystallization; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism*; D005609:Free Radicals; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008957:Models, Genetic; D009711:Nucleotides / Q000737:chemistry; D011487:Protein Conformation; D000099270:Y-Family DNA Polymerases
D000255:Adenosine Triphosphate; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D005609:Free Radicals; D006147:Guanine; D009711:Nucleotides; C453560:7,8-dihydro-8-oxoguanine; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases