Human DNA polymerase ι incorporates dCTP opposite template G via a G.C+ hoogsteen base pair

Human DNA polymerase ι (hPolι), a member of the Y family of DNA polymerases, differs in remarkable ways from other DNA polymerases, incorporating correct nucleotides opposite template purines with a much higher efficiency and fidelity than opposite template pyrimidines. We present here the crystal structure of hPolι bound to template G and incoming dCTP, which reveals a G.C+ Hoogsteen base pair in a DNA polymerase active site. We show that the hPolι active site has evolved to favor Hoogsteen base pairing, wherein the template sugar is fixed in a cavity that reduces the C1′-C1′ distance across the nascent base pair from ∼10.5 Å in other DNA polymerases to 8.6 Å in hPolι. The rotation of G from anti to syn is then largely in response to this curtailed C1′-C1′ distance. A G.C+ Hoogsteen base pair suggests a specific mechanism for hPolι's ability to bypass N2-adducted guanines that obstruct replication.