Crystal structure of the human Polϵ B-subunit in complex with the C-terminal domain of the catalytic subunit

Abstract

The eukaryotic B-family DNA polymerases include four members: Polα, Polδ, Polϵ, and Polζ, which share common architectural features, such as the exonuclease/polymerase and C-terminal domains (CTDs) of catalytic subunits bound to indispensable B-subunits, which serve as scaffolds that mediate interactions with other components of the replication machinery. Crystal structures for the B-subunits of Polα and Polδ/Polζ have been reported: the former within the primosome and separately with CTD and the latter with the N-terminal domain of the C-subunit. Here we present the crystal structure of the human Polϵ B-subunit (p59) in complex with CTD of the catalytic subunit (p261). The structure revealed a well defined electron density for p261 and the phosphodiesterase and oligonucleotide/oligosaccharide-binding domains of p59. However, electron density was missing for the p59 N-terminal domain and for the linker connecting it to the phosphodiesterase domain. Similar to Polα, p261 of Polϵ contains a three-helix bundle in the middle and zinc-binding modules on each side. Intersubunit interactions involving 11 hydrogen bonds and numerous hydrophobic contacts account for stable complex formation with a buried surface area of 3094 Å Comparative structural analysis of p59-p261 with the corresponding Polα complex revealed significant differences between the B-subunits and CTDs, as well as their interaction interfaces. The B-subunit of Polδ/Polζ also substantially differs from B-subunits of either Polα or Polϵ. This work provides a structural basis to explain biochemical and genetic data on the importance of B-subunit integrity in replisome function .

Structure of the processive human Pol δ holoenzyme

“…2c, d). Interestingly, the CTDs of Pol α and ε form equivalent complexes with their respective B-subunits but are larger and only coordinate divalent Zn 2+ ions [33][34][35][36][37] . The different size of the CTD and orientation relative to the B-subunit may correlate with the distinct functional dependencies of the three polymerases on PCNA ( Supplementary Fig.…”

Structure of DNA-CMG-Pol epsilon elucidates the roles of the non-catalytic polymerase modules in the eukaryotic replisome

“…1 - 2 ). To interpret the map, we have docked a recently published structure of human C-terminal Dpb2 bound to the Pol2 C-terminal zinc-finger appendix 34 , providing an unambiguous fit. A yeast homology model based on the human structure was subsequently used as a template for real-space refinement (Fig.…”

“…Pol2 is the catalytic subunit, with the N-terminal half containing DNA synthesis/exonuclease functions 32 . The C-terminal half of Pol2 (C-Pol2) has been predicted to contain a second polymerase fold, which has become inactivated during evolution 33 , and is followed by a zinc-finger appendix 34 . Notably, the catalytic domain of Pol epsilon is dispensable for viability (though cells are sick), while the non-catalytic C-Pol2 is essential 35 , 36 .…”

“…Notably, the catalytic domain of Pol epsilon is dispensable for viability (though cells are sick), while the non-catalytic C-Pol2 is essential 35 , 36 . Dpb2, the second largest subunit of Pol epsilon, is also essential for viability and contains an inactivated calcineurin-like exonuclease fold 34 decorated by an N-terminal appendix structurally related to the AAA+ ATPase lid domain 37 . It is clear that non-catalytic modules in Pol epsilon are required for helicase activation and origin firing 13 , 35 , 38 , although the molecular basis is poorly understood.…”

Activity and fidelity of human DNA polymerase α depend on primer structure

“…In addition, the crystal structures of the CTD-B-subunit complexes are known for yeast and human Pol␣ (11,12). With regard to the catalytic subunits of Pol␦ and Pol⑀, only the structures of the ternary complexes have been determined for their catalytic domains (22,26,42) as well as the structure of the Pol⑀ CTD-B-subunit complex (43).…”