Tidying up loose ends: the role of polynucleotide kinase/phosphatase in DNA strand break repair

Abstract

The termini of DNA strand breaks induced by internal and external factors often require processing before missing nucleotides can be replaced by DNA polymerases and the strands rejoined by DNA ligases. Polynucleotide kinase/phosphatase (PNKP) serves a crucial role in the repair of DNA strand breaks by catalyzing the restoration of 5'-phosphate and 3'-hydroxyl termini. It participates in several DNA repair pathways through interactions with other DNA repair proteins, notably XRCC1 and XRCC4. Recent studies have highlighted the physiological importance of PNKP in maintaining the genomic stability of normal tissues, particularly developing neural cells, as well as enhancing the resistance of cancer cells to genotoxic therapeutic agents.

Figure 1

Structure of Mammalian PNKP. Structural investigations have revealed the overall architecture of the PNKP catalytic domain, its interactions with kinase substrates, and its interactions with phosphorylated target proteins via its forkhead-associated (FHA) domain. (a) Overview of the catalytic domain of murine PNKP [21]. The kinase domain is orange with regions critical for catalysis (the P loop and Asp396) both highlighted. The phosphatase domain is blue with the catalytic Asp170 and Asp 172 highlighted. (b) A surface view of the murine PNKP catalytic domain in the same orientation as in (a), highlighting possible modes of substrate recognition. A minimal kinase substrate containing a 11-bp stem and a 5-nucleotide single-stranded 3'-overhang is shown bound to the catalytic domain as indicated by a combined SAXS-mutagenesis-molecular modeling study [24]. The position of the substrate 5'-OH is indicated by a yellow sphere. Two narrow channels that provide entrance to the phosphatase active site are indicated by arrows. (c) Overview of the structure of the human PNKP FHA domain bound to a doubly phosphorylated phosphopeptide derived from XRCC1 [28]. The FHA domain is in blue, the XRCC1 phosphopeptide in white sticks. (d) Amino acid sequence alignment of the regions of human (hu) and murine (mu) XRCC1 and XRCC4 bound by the PNKP FHA domain. Sites of CK2 phosphorylation are highlighted in purple, conserved acidic residues are in red, and conserved residues at −4 and +3 that also are selectively recognized by the FHA are colored green. (e) Detailed view of PNKP FHA–phospho-XRCC1 interactions. Residues contributing to key interactions are shown as sticks, with hydrogen bonding and salt bridging interactions indicated by yellow dotted lines. Note the presence of multiple conformers of pSer at the −1 position [28].

Figure 2

Model of XRCC4 and PNKP participation in NHEJ. The model represents a double strand break with 5'-OH termini. After assembly of the DNA-PK complex composed of the Ku70–Ku80 heterodimer (purple) and DNA-PK_cs (light blue) at the strand break termini, XRCC4 (green) is recruited along with PNKP (yellow). (Identical protein recruitment and enzymatic activity would occur at each break terminus, but only one is shown for clarity.) Upon completion of phosphorylation of both termini, phosphorylated XRCC4–DNA ligase IV (green–dark blue) complex binds PNKP through the FHA domain and thereby displaces PNKP and non-phosphorylated XRCC4 from the strand break termini, so that other proteins, including DNA ligase IV, can gain access to the termini to complete the repair.

Figure 3

Participation of PNKP in DNA repair pathways. The figure highlights the requirement of PNKP to process strand break termini at SSBs directly introduced by DNA damaging agents (a and b), at SSBs introduced in the course of BER (c and d), and at directly induced DSBs (e–g). (a) PNKP hydrolyzes 3'-phosphate and phosphorylates 5'-OH termini at a radiation-induced SSB prior to replacement of the missing nucleotide by DNA polymerase β and strand rejoining by DNA ligase III (LIG3). (b) Strand rejoining at a topoisomerase 1 (TopoI)–DNA “dead-end” complex first requires the hydrolysis of the covalent bond attaching topoisomerase I to the DNA 3'-phosphate group, followed by PNKP-mediated correction of the 3'-phosphate and 5'-OH termini and strand rejoining by DNA ligase III. (c) BER by DNA glycosylases that possess β,δ-AP lyase activity, such as NEIL1 and NEIL2, remove oxidized bases and cleave the abasic sites, leaving 3'-phosphate groups that are acted upon by PNKP. (d) DNA glycosylases that have no AP lyase activity, such as methylpurine-DNA glycosylase (MPG), create abasic sites that can be acted upon by NEIL1 or NEIL2, thereby generating strand breaks with 3'-phosphate termini. (e) The NHEJ pathway for DSB repair involves end-binding by the Ku heterodimer and DNA-PK_cs followed by end processing by PNKP (or other proteins, such as Artemis) and strand rejoining by DNA ligase IV (LIG4), which exists in complex with XRCC4 and possibly XLF (see Fig. 2 for a more detailed model of PNKP involvement in 5'-phosphorylation at DSB termini). (f) Alternative, XRCC1-dependent, DSB repair pathway. (g) Removal of phosphoglycolate groups at 3'-overhanging DSB termini entails hydrolysis of the glycolate group by TDP1 followed by dephosphorylation by PNKP as a component of the NHEJ pathway.

Box 1, Figure I

Structures of DNA strand break termini requiring processing prior to ligation. (a) Hydroxyl radical attack of the deoxyribose can generate 5'-hydroxyl termini and 3'-phosphate (i) or 3'-phosphoglycolate (ii). (b) Bleomycin-induced DSB with 3'-phosphoglycolate termini. (c) Topoisomerase I (TopoI)–DNA “dead-end” complex generated by camptothecin. The enzyme, which is covalently attached to the DNA 3'-phosphate through a tyrosine residue, can be released from the DNA by TDP1. (d) DNA glycosylases with AP lyase activity cleave abasic sites. The products depend on the cleavage chemistry: β-elimination leads to the 4-hydroxy-2-pentenal moiety bound to the 3'-phosphate (i), whereas βδ-elimination produces a 3'-phosphate (ii). Phosphate groups are represented by the letter P.