Structural basis for the phosphatase activity of polynucleotide kinase/phosphatase on single- and double-stranded DNA substrates

Abstract

Polynucleotide kinase/phosphatase (PNKP) is a critical mammalian DNA repair enzyme that generates 5′-phosphate and 3′-hydroxyl groups at damaged DNA termini that are required for subsequent processing by DNA ligases and polymerases. The PNKP phosphatase domain recognizes 3′-phosphate termini within DNA nicks, gaps, or at double-or single-strand breaks. Here we present a mechanistic rationale for the recognition of damaged DNA termini by the PNKP phosphatase domain. The crystal structures of PNKP bound to single-stranded DNA substrates reveals a narrow active site cleft that accommodates a single-stranded substrate in a sequence-independent manner. Biochemical studies suggest that the terminal base pairs of double-stranded substrates near the 3′-phosphate are destabilized by PNKP to allow substrate access to the active site. A positively charged surface distinct from the active site specifically facilitates interactions with double-stranded substrates, providing a complex DNA binding surface that enables the recognition of diverse substrates.DNA substrate recognition | HAD family | | protein-DNA complexes | X-ray crystallography D NA damage induced by ionizing radiation, as well as DNA repair intermediates generated during base excision repair, often result in the formation of DNA ends containing 3′-phosphate/5′-hydroxyl termini. Because DNA polymerases and ligases require 3′-hydroxyl/5′-phosphate termini to complete DNA repair, cells have evolved enzymes to recognize and process these damaged DNA ends. Mammalian polynucleotide kinase/phosphatase (PNKP) contains 3′-phosphatase and 5′-kinase activities present in two distinct active sites (1). The critical role of PNKP in various DNA repair processes is underlined by the fact that RNAi knockdown of PNKP leads to marked sensitization of human cells to spontaneous mutation as well as to various genotoxic agents (2). A particularly important role is attributed to the phosphatase activity as small molecules that selectively inhibit the phosphatase but not the kinase activity of PNKP sensitize human cells to DNA damage in a PNKP-dependent manner (3, 4). Mutations in the PNKP phosphatase domain are associated with the developmental neurological disorder MCSZ, revealing a critical role for PNKP in human neurological development (5).The PNKP phosphatase domain plays a central role in several critical DNA repair processes. For example, PNKP participates in base excision repair (BER) in partnership with the NEIL (endonuclease VIII-like) DNA glycosylases. NEILs exhibit β,δ-AP (aminopurine) lyase activity, which produces single nucleotide gap DNAs containing 3′-phosphate termini that are subsequently dephosphorylated by PNKP prior to gap filling and ligation (6, 7). PNKP is also associated with the repair of double-strand breaks through the nonhomologous end joining (NHEJ) pathway (8, 9), in a manner that relies on the interaction of PNKP with the NHEJ scaffolding protein, XRCC4 (10). Consistent with its role in these DNA repair pathways, PNKP recognizes 3′-phosphate termini ...

Rewriting the rules for end joining via enzymatic splicing of DNA 3′-PO 4 and 5′-OH ends

“…Nonligatable 3′-PO 4 ends are also generated during base excision repair catalyzed by DNA glycosylase/lyase enzymes, during the repair of trapped covalent topoisomerase IB-DNA adducts by tyrosyl-DNA phosphodiesterase 1, and during DNA damage inflicted by ionizing radiation. One way nature solves this "dirty end" problem is by deploying a variety of "end healing" enzymes (8)(9)(10)(11)(12)(13)(14). These include 3′-phosphoesterases that convert a 3′-PO 4 to a 3′-OH and 5′-kinases that transform a 5′-OH to a 5′-PO 4 , thereby enabling break sealing by the classic ligase pathway.…”

DNA end processing by polynucleotide kinase/phosphatase

“…Thus, intimate coupling of the kinase and phosphatase activities may have important implications for controlling the orderly progression of DNA end processing. Coquelle et al (5) posit that productive engagement of a 3′-PO 4 Coquelle et al posit that productive engagement of a 3′-PO 4 terminus may block access of a 5′-OH to the kinase active site.…”

“…For instance, how are PNK activities regulated? The observation of DNA-induced fit conformational changes (5,12), interdomain kinase/phosphatase flexibility (7), and PNK posttranslational modifications such as phosphorylation (15,16) suggests multiple avenues for modulating PNK activity. PNK is further integrated into multienzyme SSB and DSB repair pathways complexes by binding scaffolding proteins such as Xrcc1 (17) and Xrcc4 (18).…”

“…To cope with structurally diverse DNA termini, eukaryotic cells have acquired an extensive array of enzymatic activities to tailor DNA ends for ligation. Now, in PNAS, key molecular insights into the mechanisms of DNA end recognition and processing are emerging (5).…”

“…From the structures, the authors hypothesize that PNK must separate the strands of the damaged dsDNA or nicked DNA duplex to promote 3′-PO 4 processing. To test this, Coquelle et al (5) use the DNA base analogue 2-aminopurine to monitor the terminal base pairing proximal to the 3′-PO 4 end in duplex DNA. Their results show that, consistent with the structural observations, PNK induces strand separation of two or three terminal base pairs proximal to the 3′-PO 4 end.…”