title: Human DNA polymerase η is pre-aligned for dNTP binding and catalysis.
abstract: Pre-steady-state kinetic studies on Y-family DNA polymerase η (Polη) have suggested that the polymerase undergoes a rate-limiting conformational change step before the phosphoryl transfer of the incoming nucleotide to the primer terminus. However, the nature of this rate-limiting conformational change step has been unclear, due in part to the lack of structural information on the Polη binary complex. We present here for the first time a crystal structure of human Polη (hPolη) in binary complex with its DNA substrate. We show that the hPolη domains move only slightly on dNTP binding and that the polymerase by and large is pre-aligned for dNTP binding and catalysis. We also show that there is no major reorientation of the DNA from a nonproductive to a productive configuration and that the active site is devoid of metals in the absence of dNTP. Together, these observations lead us to suggest that the rate-limiting conformational change step in the Polη replication cycle likely corresponds to a rate-limiting entry of catalytic metals in the active site.
authors.affiliation: Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, NY 10029, USA.
authors.forename: Ajay
authors.initials: A
authors.lastname: Ummat
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Silverstein
authors.forename: Rinku
authors.initials: R
authors.lastname: Jain
authors.forename: Angeliki
authors.initials: A
authors.lastname: Buku
authors.forename: Robert E
authors.initials: RE
authors.lastname: Johnson
authors.forename: Louise
authors.initials: L
authors.lastname: Prakash
authors.forename: Satya
authors.initials: S
authors.lastname: Prakash
authors.forename: Aneel K
authors.initials: AK
authors.lastname: Aggarwal
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D003854:Deoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D000099270:Y-Family DNA Polymerases
chemical_list: D004259:DNA-Directed DNA Polymerase; D003854:Deoxyribonucleotides; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases