Separate roles of structured and unstructured regions of Y-family DNA polymerases

Abstract

All organisms have multiple DNA polymerases specialized for translesion DNA synthesis (TLS) on damaged DNA templates. Mammalian TLS DNA polymerases include Pol eta, Pol iota, Pol kappa, and Rev1 (all classified as "Y-family" members) and Pol zeta (a "B-family" member). Y-family DNA polymerases have highly structured catalytic domains; however, some of these proteins adopt different structures when bound to DNA (such as archaeal Dpo4 and human Pol kappa), while others maintain similar structures independently of DNA binding (such as archaeal Dbh and Saccharomyces cerevisiae Pol eta). DNA binding-induced structural conversions of TLS polymerases depend on flexible regions present within the catalytic domains. In contrast, noncatalytic regions of Y-family proteins, which contain multiple domains and motifs for interactions with other proteins, are predicted to be mostly unstructured, except for short regions corresponding to ubiquitin-binding domains. In this review we discuss how the organization of structured and unstructured regions in TLS polymerases is relevant to their regulation and function during lesion bypass.

Figure 1. Catalytic domain structures of Y-family polymerases

All molecular graphics images were prepared using MacPyMOL (http://www.pymol.org/), based on the PDB entries indicated in the parenthesis. Y-family proteins are rainbow-colored; N-terminal regions are shown in blue and C-terminal regions in red. A. Dpo4+DNA (1JX4), B. Apo-Dpo4 (2RDI), C. Dpo4+PCNA1 (3FDS), D. Apo-hPol κ (1T94), E. hPol κ+DNA+TTP (2OH2), F. scPol η+DNA+dCTP (2R8J), G. hPol ι+DNA+dGTP (3GV8), H. hRev1+DNA+dCTP (3GQC). In H, some portions of hRev1 around the template G and the incoming dCTP are enlarged. When truncated forms were used for structural analysis, the regions analyzed are denoted in the parenthesis below each of the proteins.

Figure 1. Catalytic domain structures of Y-family polymerases

All molecular graphics images were prepared using MacPyMOL (http://www.pymol.org/), based on the PDB entries indicated in the parenthesis. Y-family proteins are rainbow-colored; N-terminal regions are shown in blue and C-terminal regions in red. A. Dpo4+DNA (1JX4), B. Apo-Dpo4 (2RDI), C. Dpo4+PCNA1 (3FDS), D. Apo-hPol κ (1T94), E. hPol κ+DNA+TTP (2OH2), F. scPol η+DNA+dCTP (2R8J), G. hPol ι+DNA+dGTP (3GV8), H. hRev1+DNA+dCTP (3GQC). In H, some portions of hRev1 around the template G and the incoming dCTP are enlarged. When truncated forms were used for structural analysis, the regions analyzed are denoted in the parenthesis below each of the proteins.

Figure 2. Disordered profile plots of Dpo4 (A), hPol κ (B), spPol κ (C) and cePol κ (D)

Each of the plots was obtained from the DISOPRED2 server (http://bioinf.ucl.ac.uk/diospred/), after inputting the entire primary sequence of the respective protein. Amino acid sequences of domains and motifs are shown below the plot, in which conserved residues are underlined.

Figure 3

Disordered profile plots of scPol η (A) and hPol η (B).

Figure 4. Yeast two-hybrid assay for binding to PCNA and hRev1-CTD

Each segment of hPol η, hPol ι, hPol κ or hRev1 was inserted into pLexA (BD) vector and the entire region of PCNA or a region (951–1251) of hRev1 was inserted into pB42AD vector. Amino acid sequences of the inserted segments are shown in the right, in which conserved residues are underlined and altered sequences are shown in Italic. “No” indicates empty vector. Experiments were performed as described previously (Ohashi et al., 2009).

Figure 5. Multiple alignment of the N-terminal regions in mammalian Pol

ι homologues. In the upper amino acid sequence alignments, the Met residues of human and mouse Pol ι that were previously assigned as the first residue are underlined. In the lower sequences, the regions from the newly assigned Met start codon to the previously assigned one in the gene coding for the 740 or 739 aa protein are shown for human Pol ι, and are derived from the NCBI entries NM_007195 and AK301578, respectively. Many cDNA clones with the 5′-end sequence identical to either one of the two entries are found in human EST libraries at almost equal frequencies, implying that the difference is due to heterogeneity, not to sequence error. CGA repeats in the newly identified N-terminal sequences are denoted by a horizontal line with an arrow.

Figure 6. Disordered profile plot of hPol ι

The entire 740 aa hPol ι sequence including the newly identified N-terminal 25 amino acids was analyzed for disorder probability, but the positions of the motifs and domains are presented using the old numbering system (reflecting the N-terminally-truncated 715 amino acid species). The old numbering may be converted to the new full-length sequence by addition of 25.

Figure 7. Disordered profile plot of hRev1

The location and sequence of a putative PIP-like sequence (1110–1117) in the C-terminal region is indicated by ‘PIP?’. However, as shown in Fig 4A, a region of Rev1 spanning the putative PIP sequence (1102–1124) showed very weak PCNA binding activity in yeast two-hybrid assay.