APOBEC-catalyzed cytosine-to-uracil deamination of single-stranded DNA (ssDNA) has beneficial functions in immunity and detrimental effects in cancer. APOBEC enzymes have intrinsic dinucleotide specificities that impart hallmark mutation signatures. Although numerous structures have been solved, mechanisms for global ssDNA recognition and local target-sequence selection remain unclear. Here we report crystal structures of human APOBEC3A and a chimera of human APOBEC3B and APOBEC3A bound to ssDNA at 3.1-A and 1.7-A resolution, respectively. These structures reveal a U-shaped DNA conformation, with the specificity-conferring -1 thymine flipped out and the target cytosine inserted deep into the zinc-coordinating active site pocket. The -1 thymine base fits into a groove between flexible loops and makes direct hydrogen bonds with the protein, accounting for the strong 5′-TC preference. These findings explain both conserved and unique properties among APOBEC family members, and they provide a basis for the rational design of inhibitors to impede the evolvability of viruses and tumors.
Bibliographical noteFunding Information:
We thank D. Largaespada and D. Yee for insightful comments, R. Moorthy for oligonucleotide sample preparations, and J. Stivers (Pharmacology and Molecular Sciences Department, Johns Hopkins University, Baltimore, Maryland, USA) for providing the human UNG2 expression construct and purification protocol. This work was supported by grants from the US National Institutes of Health (NIGMS R01-GM118000 to R.S.H. and H.A., NIGMS R35-GM118047 to H.A., NIGMS R01-GM110129 to D.A.H., NCI R21-CA206309 to R.S.H., and DP2-OD007237 and NIGMS P41-GM103426 to R.E.A.), the NSF (CHE060073N to R.E.A.), the Prospect Creek Foundation (R.S.H. and D.A.H.), and the University of Minnesota Masonic Cancer Center (Spore-Program-Project-Planning Seed Grant to R.S.H.). This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the US National Institutes of Health (NIGMS P41-GM103403). The Pilatus 6M detector on the 24-ID-C beamline is funded by an NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. R.S.H. is supported as the Margaret Harvey Schering Land Grant Chair for Cancer Research and as an Investigator of the Howard Hughes Medical Institute
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