Structural basis for recruitment of translesion DNA polymerase Pol IV/DinB to the beta-clamp

Abstract

Y-family DNA polymerases can extend primer strands across template strand lesions that stall replicative polymerases. The poor processivity and fidelity of these enzymes, key to their biological role, requires that their access to the primer-template junction is both facilitated and regulated in order to minimize mutations. These features are believed to be provided by interaction with processivity factors, beta-clamp or proliferating cell nuclear antigen (PCNA), which are also essential for the function of replicative DNA polymerases. The basis for this interaction is revealed by the crystal structure of the complex between the 'little finger' domain of the Y-family DNA polymerase Pol IV and the beta-clamp processivity factor, both from Escherichia coli. The main interaction involves a C-terminal peptide of Pol IV, and is similar to interactions seen between isolated peptides and other processivity factors. However, this first structure of an entire domain of a binding partner with an assembled clamp reveals a substantial secondary interface, which maintains the polymerase in an inactive orientation, and may regulate the switch between replicative and Y-family DNA polymerases in response to a template strand lesion.

Fig. 1. β-clamp–Pol IV-LF complex. (A) Secondary structure cartoon of the β-clamp dimer (magenta and yellow) with a Pol IV LF domain bound on each side, superimposed on the solvent-accessible surface of the complex. The two halves of the β-clamp and the two LF domains are related by a non-crystallographic 2-fold axis running perpendicular to the plane of view. [This and all other molecular graphics were generated with PyMOL (DeLano Scientific LLC, www.delanoscientific.com).] (B) Superimposition of the LF domain of Pol IV (red tube) and the equivalent structures from S.solfaraticus Dpo4 (cyan), S.solfataricus Dbh (yellow) and yeast Pol η (green). (C) As (B) but rotated 90° around the horizontal.

Fig. 2. Binding of Pol IV C-terminal peptide to the β-clamp. (A) The C-terminal peptide of Pol IV from residues 341 to 351 (stick model) binds into a channel on the surface of the β-clamp, with the side chains of Met343, Leu347, Leu349 and Leu351 inserted into hydrophobic pockets in the base of the channel. The molecular surface of the β-clamp is coloured according to the residue type: acidic, red; basic, blue; polar, orange; hydrophobic, green. (B) View perpendicular to (A) showing residues from the β-clamp involved in interaction with the Pol IV C-terminus.

Fig. 3. Protein–protein interface. (A) Stereo pair of the Pol IV LF domain (blue cartoon) binding at the interface between the two monomers of the β-clamp (yellow/magenta surfaces). Pol IV residues involved in direct interaction with the β-clamp are shown as sticks. The C-terminal peptide (see text) runs off to the left in this view. (B) Close-up stereo pair showing details of the protein–protein interaface between Pol IV-LF and the β-clamp. The core of the interaction is provided by the insertion of a hydrophobic ‘peg’ provided by the side chain of Leu98 of the β-clamp into a hydrophobic ‘hole’ formed by the side chains of Val303, Trp304 and Pro305 of Pol IV. This is surrounded by a ring of polar and electrostatic and solvent-bridged interactions between Pol IV and both monomers of the β-clamp. The view is from the opposite face of the β-clamp ring to that in (A).

Fig. 4. Comparison of β-clamp interactions with Pol IV-LF and the clamp loader δ-subunit. (A) Superposition of the clamp loader δ-subunit–β-clamp complex (pink, green) with one half of the Pol IV-LF–β-clamp complex (blue, gold) based on a least squares fit of the Cα positions in the common β-clamp subunit component. The β-clamp-binding segment of the δ-subunit is highlighted in red. (B) Close-up of the superposed β-clamp-binding peptide segments of the clamp loader δ-subunit (pink carbons) and Pol IV-LF (blue carbons). There is a one-to-one correspondence in conformation and composition except for the extreme C-terminus of Pol IV, where the LGL tri-peptide replaces the LF dipeptide of the δ-subunit.

Fig. 5. Binding site conservation in processivity clamp interactions. (A) Secondary structure cartoon of the E.coli β-clamp–Pol IV-LF complex compared with other processivity clamp–peptide complexes. (B) Human PCNA–p21^cip/waf peptide. (C) Pyrococcus furiosus PCNA–replication factor C peptide. (D) Phage RB69 sliding clamp–DNA polymerase. Despite the very low level of sequence similarity between the bacterial, mammalian, archaeal and viral clamps, the gross topology of the peptide-binding site is conserved in all the systems.

Fig. 6. The Pol IV/DinB ‘polymerase switch’. (A) Model of a Pol IV/DinB type Y-class polymerase (red) bound to the β-clamp (gold) in the ‘locked-down’ inactive position. The position of the polymerase was modelled by superimposing the LF domain of the archaeal Dpo4 enzyme from the DNA complex (PDB code: 1JXL) onto the Pol IV-LF in the complex with the β-clamp described here (blue). Modelled in this position, the polymerase makes no steric clashes with the β-clamp, but cannot access the primer–template junction of the DNA (primer strand, pink, template strand, green). Furthermore, the face of the β-clamp is not obstructed, so that a replicative DNA polymerase bound to the DNA could be accommodated. (B) Model of a Pol IV/DinB type Y-class polymerase bound to the primer–template junction following a polymerase switch. The position of the polymerase was modelled by superimposing the DNA from the Dpo4–DNA complex onto the end of a long DNA molecule running perpendicularly through the lumen of the clamp. Disruption of the substantial protein–protein interface between the clamp and the polymerase in the ‘locked-down’ conformation would provide a barrier to attainment of this active complex, disfavouring access by the translesion polymerase except when the primer–template junction is vacated for a long period. Contact with the clamp is maintained by the C-terminal clamp-binding peptide (blue), which tethers the enzyme to the replication complex.