Human DNA Polymerase η Is Pre-Aligned for dNTP Binding and Catalysis

• Published in: Journal of molecular biology Vol. 415; no. 4; pp. 627 - 634
• Main Authors: Ummat, Ajay, Silverstein, Timothy D., Jain, Rinku, Buku, Angeliki, Johnson, Robert E., Prakash, Louise, Prakash, Satya, Aggarwal, Aneel K.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 27.01.2012
• Subjects: binding and catalysis; Binding Sites; Catalysis; catalytic activity; crystal structure; Crystallography, X-Ray; Deoxyribonucleotides - chemistry; Deoxyribonucleotides - metabolism; DNA; DNA polymerase; DNA repair; DNA-directed DNA polymerase; DNA-Directed DNA Polymerase - chemistry; DNA-Directed DNA Polymerase - genetics; DNA-Directed DNA Polymerase - metabolism; DNA-Directed DNA Polymerase - physiology; Humans; metals; Models, Biological; Models, Molecular; Mutagenesis, Site-Directed; Protein Binding; Protein Structure, Quaternary; Protein Structure, Secondary; translesion DNA synthesis; Y-family DNA polymerase; PAD; translesion DNA synthesis; DNA polymerase; MR; binding and catalysis; yPolη; hPolη; DNA repair; polη; Y-family DNA polymerase
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2011.11.038

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. [Display omitted] ► For the first time, a structure of the tumor suppressor hPolη in binary complex with DNA is presented. ► Surprisingly, the polymerase is pre-aligned for dNTP binding and catalysis. ► There was no reorientation of the DNA from a nonproductive to a productive configuration. ► The active site is devoid of metals in the absence of dNTP. ► The replication cycle is likely limited by the rate-limiting entry of metals in the active site.