Structural basis for novel interactions between human translesion synthesis polymerases and proliferating cell nuclear antigen

Abstract

Translesion synthesis (TLS) is a DNA damage tolerance mechanism that allows continued DNA synthesis, even in the presence of damaged DNA templates. Mammals have multiple DNA polymerases specialized for TLS, including Poleta, Poliota, and Polkappa. These enzymes show preferential bypass for different lesions. Proliferating cell nuclear antigen (PCNA), which functions as a sliding clamp for the replicative polymerase Poldelta, also interacts with the three TLS polymerases. Although many PCNA-binding proteins have a highly conserved sequence termed the PCNA-interacting protein box (PIP-box), Poleta, Poliota, and Polkappa have a noncanonical PIP-box sequence. In response to DNA damage, Lys-164 of PCNA undergoes ubiquitination by the RAD6-RAD18 complex, and the ubiquitination is considered to facilitate TLS. Consistent with this, these three TLS polymerases have one or two ubiquitin binding domains and are recruited to replication forks via interactions with ubiquitinated PCNA involving the noncanonical PIP-box and ubiquitin binding domain. However, it is unclear how these TLS polymerases interact with PCNA. To address the structural basis for interactions between different TLS polymerases and PCNA, we determined crystal structures of PCNA bound to peptides containing the noncanonical PIP-box of these polymerases. We show that the three PIP-box peptides interact with PCNA in different ways, both from one another and from canonical PIP-box peptides. Especially, the PIP-box of Poliota adopts a novel structure. Furthermore, these structures enable us to speculate how these TLS polymerases interact with Lys-164-monoubiquitinated PCNA. Our results will provide clues to understanding the mechanism of preferential recruitment of TLS polymerases to the stalled forks.

FIGURE 1.

A, schematic features of the primary structures of human Polη, Polκ, and Polι. Polη has a UBD termed the UBZ domain, which is a CCHH-type zinc finger domain. Polκ has two UBZ domains, which are CCHC-type zinc finger domains. Polι has two UBDs termed the UBM domains. B, sequence alignment of PCNA-interacting regions that include canonical and noncanonical PIP-boxes. The box delineates the eight residues of canonical and noncanonical PIP-boxes, which are numbered as shown above the canonical sequence. The canonical PIP-box elements at positions 1, 4, 7, and 8 are shown on a black background. Acidic residues conserved at p5 of the three TLS polymerases are shown on a gray background. The conserved methionines of Polη, the basic residues of Polκ and Polι at p1, and the leucine of Polι at p8 are shown on a light gray background. Asterisks indicate the C termini of the proteins, and numbers in parentheses are the total number of residues in the full-length proteins.

FIGURE 2.

Structure of PCNA bound to the Polη (A), Polκ (B), or Polι (C) peptide. Overall structures in the asymmetric units are shown by ribbon representations (left). TLS polymerase peptides are shown in yellow. In A, the IDCL in the A-molecule is shown by a thick coil. The black spheres in B are zinc ions from the crystallization buffer. The interactions of the U-molecules of Polη, Polκ, and Polι with the A-molecule of PCNA (blue) are shown as representative structures by stereo pairs (right). For clarity, the noncanonical PIP-boxes are colored orange. The regions preceding and following the PIP-boxes are shown in yellow. Electrostatic interactions are indicated by orange dots.

FIGURE 3.

Hydrophobic plug-socket interaction of the Polη (A) or Polι (B) peptide with PCNA and superimposition of PIP-box structures bound to PCNA (C). A and B, PCNA is shown by a surface model, and residues of PCNA that interact with the three-forked hydrophobic plug are colored gray. PIP-box residues are shown by stick models. Residues of PCNA that interact with the Met-701 (p1) residue of Polη in the Q-pocket are colored light pink. C, structures of p21, Polη, Polκ, and Polι bound to PCNA are shown in light blue, pink, green, and yellow, respectively. PIP-box residues are shown by stick models, and only some PIP-box residues of Polι are denoted.

FIGURE 4.

Physical interactions of Polη and Polι with PCNA. GST-fused TLS polymerase (GST-Polη or GST-Polι) was mixed with WT or H44A mutant PCNA (lanes 1–4). As controls, GST or buffer was mixed with PCNA (WT or H44A mutant) (lanes 5–8). Samples were bound to glutathione-Sepharose beads, which were then washed and eluted by glutathione-containing buffer. Eluents were analyzed by Western blotting. Inputs of WT and H44A are also shown in lanes 9 and 10, respectively.

FIGURE 5.

Proposed models of the interaction of Lys-164-monoubiquitinated PCNA with Polη or Polκ (A) and Polι (B). The ubiquitin moieties linked to Lys-164 are shown by ellipsoids. N- and C-terminal sides of TLS polymerase fragments are indicated by N and C, respectively.