An overview of Y-Family DNA polymerases and a case study of human DNA polymerase η

Abstract

Y-Family DNA polymerases specialize in translesion synthesis, bypassing damaged bases that would otherwise block the normal progression of replication forks. Y-Family polymerases have unique structural features that allow them to bind damaged DNA and use a modified template base to direct nucleotide incorporation. Each Y-Family polymerase is unique and has different preferences for lesions to bypass and for dNTPs to incorporate. Y-Family polymerases are also characterized by a low catalytic efficiency, a low processivity, and a low fidelity on normal DNA. Recruitment of these specialized polymerases to replication forks is therefore regulated. The catalytic center of the Y-Family polymerases is highly conserved and homologous to that of high-fidelity and high-processivity DNA replicases. In this review, structural differences between Y-Family and A- and B-Family polymerases are compared and correlated with their functional differences. A time-resolved X-ray crystallographic study of the DNA synthesis reaction catalyzed by the Y-Family DNA polymerase human polymerase η revealed transient elements that led to the nucleotidyl-transfer reaction.

Figure 1

Diagram of domain structures and protein interactions of Y-Family polymerases. (A) Catalytic and regulatory regions of six subgroups of Y-Family polymerases. The catalytic core contains finger, palm, and thumb subdomains. LF denotes the little finger domain. PIP stands for the PCNA interaction peptide, RIR for the Rev1 interaction region, NLS for the nuclear localization signal, Ub for ubiquitin, UBZ for the Ub-binding zinc finger, UBM for the Ub-binding module, and BRCT for the BRCA1 C-terminal domain. (B) Schematic diagram of ubiquitinated PCNA interacting with all Y-Family polymerases and Rev1 interacting with pol η, ι, and κ.

Figure 2

Varied interactions between the catalytic core (CC) and little finger domain (LF) of Y-Family polymerases. All structures shown here are of ternary complexes with DNA and dNTP. The CCs are superimposed, and the DNA and dNTP have been removed for clarity. (A) The relative orientation of CC (blue) and LF (purple) differs in archaeal Dbh [Protein Data Bank (PDB) entry 3BQ1] and Dpo4 (PDB entry 2AGQ), resulting a small gap between the CC and LF in Dpo4 and a large opening in Dbh. (B) The gap between the CC and LF is large in pol κ (PDB entry 2OH2) and Rev1 (PDB entry 2AQ4) and small to nonexistent in pol η (PDB entry 4ED8) and ι (PDB entry 3GV8). For the integrity of the catalytic region, pol κ uses its N-terminal extension (N-clasp, colored yellow) to bridge the CC and LF (in the back for this view), and Rev1 uses the N-terminal extension to fill the gap (crossing from the back to the front).

Figure 3

Structural domains in human pol η. (A) Diagram of the linear arrangement of functional domains in human pol η. Domains are color-coded. (B) Crystal structure of the catalytic region (amino acids 1–432) in a complex with DNA and dNTP (PDB entry 3MR2). (C) Structural model of the quaternary complex of pol η RIR complexed with the Rev1 CTD, Rev3, and Rev7. The model is a composite of RIR (η)–Rev1 (PDB entry 2LSK) and RIR(κ)–Rev1–Rev3–Rev7 complexes (PDB entry 4GK5). (D) NMR structure of the UBZ domain (PDB entry 2I5O) with the residues interacting with Ub shown as sticks. The Zn²⁺ ion is shown as a green sphere. (E) Crystal structure of human PCNA complexed with the pol η PIP (PDB entry 2ZVK). All parts of human pol η are shown as ribbon diagrams, and their interacting partners are shown as a molecular surface. The α-helices of pol η are shown as cylinders in panels B and E.

Figure 4

Different conformational changes in replicases and Y-Family polymerases. (A and B) Open (PDB entry 4BDP) and closed (PDB entry 3THV) structures, respectively, of Bacillus DNA polymerase I. The structure of the apo polymerase is identical to the structure of a polymerase–DNA binary complex, and both have an open conformation. The “O” helix (colored deep purple) and the two surrounding helices (colored pink) undergo a “closing” motion upon binding of a correct incoming dNTP and metal ion (shown as a green sphere). (C and D) Structures of Dpo4 in apo (PDB entry 2RDI) and DNA-bound (PDB entry 2AGQ) forms, respectively. The little finger domain (LF) rotates nearly 130° between the two forms.

Figure 5

Comparison of the active site of a replicase and pol η. Close-up views of the ternary complexes of (A) T7 DNA polymerase (PDB entry 1T7P) and (B) pol η (PDB entry 3MR3). The finger (blue), palm (red), thumb (green), and LF (purple) domains are shown as ribbon diagrams superimposed with semitransparent molecular surfaces. DNA is shown as yellow (primer) and orange (template) tube and ladders; incoming dNTPs are shown as white sticks, and metal ions are shown as cyan spheres. The template base and catalytic metal ions in the T7 complex are barely visible, while the cis-syn thymine dimer (shown as red sticks), incoming nucleotide, and both active site metal ions in the pol η complex are solvent-exposed.

Figure 6

Nucleotidyl-transfer reaction. (A) Diagram of the reaction catalyzed by DNA polymerases. (B) Time-lapse recording of the reaction catalyzed by human pol η. At time zero, a Ca²⁺ ion occupied the B site, and the 3′ end of the primer (circled) is not aligned with the α-phosphate of dNTP. At 40 s (40 s after the addition of Mg²⁺), both metal ion-binding sites A and B become occupied by Mg²⁺ ions, and the 3′-OH and α-phosphate are aligned. However, no chemical reaction is detected. At 80 s, a transient water appears (indicated by a gray arrowhead), and the new bond starts to form as indicated by the black double arrow. At 230 s, there are more products than substrate, and the third Mg²⁺ ion partially occupies the C site. (C) Composite of mixed substrate (colored yellow) and product (colored blue) in the middle of the reaction time course.