Mechanism of DNA substrate recognition by the mammalian DNA repair enzyme, Polynucleotide Kinase

Bernstein, Nina; Hammel, Michal; Mani, Rajam S.; Weinfeld, Michael; Pelikán, Martin; Tainer, John A.; Glover, J. N. Mark

doi:10.1093/nar/gkp597

Cited by 50 publications

(49 citation statements)

References 45 publications

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“…19 Additional proteins also contribute to end processing, including polynucleotide kinase 39 phosphatase. 20 The structure-specific nuclease, Artemis, is also required for rejoining a subset of DNA ends, which appear to represent those that incur some level of resection, possibly owing to their increased complexity. 21 Overall, NHEJ represents a compact process, with current evidence suggesting that only a single Ku molecule binds to each end ( Figure 1).…”

Section: An Overview Of Non-homologous End-joining and Homologous Recmentioning

confidence: 99%

DNA DSB repair pathway choice: an orchestrated handover mechanism

Kakarougkas

Jeggo²

2014

BJR

237

186

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Cite this article as: Kakarougkas A, Jeggo PA. DNA DSB repair pathway choice: an orchestrated handover mechanism. Br J Radiol 2014;87:20130685. RADIOBIOLOGY SPECIAL FEATURE: REVIEW ARTICLE DNA DSB repair pathway choice: an orchestrated handover mechanism A KAKAROUGKAS, MSc, PhD, and P A JEGGO, PhD Genome Damage and Stability Centre, University of Sussex, Brighton, UK Address correspondence to: Professor Penny Jeggo E-mail: p.a.jeggo@sussex.ac.uk ABSTRACT DNA double strand breaks (DSBs) are potential lethal lesions but can also lead to chromosome rearrangements, a step promoting carcinogenesis. DNA non-homologous end-joining (NHEJ) is the major DSB rejoining process and occurs in all cell cycle stages. Homologous recombination (HR) can additionally function to repair irradiation-induced two-ended DSBs in G2 phase. In mammalian cells, HR predominantly uses a sister chromatid as a template for DSB repair; thus HR functions only in late S/G2 phase. Here, we review current insight into the interplay between HR and NHEJ in G2 phase. We argue that NHEJ represents the first choice pathway, repairing approximately 80% of X-ray-induced DSBs with rapid kinetics. However, a subset of DSBs undergoes end resection and repair by HR. 53BP1 restricts resection, thereby promoting NHEJ. During the switch from NHEJ to HR, 53BP1 is repositioned to the periphery of enlarged irradiation-induced foci (IRIF) via a BRCA1-dependent process. K63-linked ubiquitin chains, which also form at IRIF, are also repositioned as well as receptor-associated protein 80 (RAP80), a ubiquitin binding protein. RAP80 repositioning requires POH1, a proteasome component. Thus, the interfacing barriers to HR, 53BP1 and RAP80 are relieved by POH1 and BRCA1, respectively. Removal of RAP80 from the IRIF core is required for loss of the ubiquitin chains and 53BP1, and for efficient replication protein A foci formation. We propose that NHEJ is used preferentially to HR because it is a compact process that does not necessitate extensive chromatin changes in the DSB vicinity.

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Section: An Overview Of Non-homologous End-joining and Homologous Recmentioning

confidence: 99%

DNA DSB repair pathway choice: an orchestrated handover mechanism

Kakarougkas

Jeggo²

2014

BJR

237

186

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“…S2). The kinase domain overhangs the phosphatase active site, in particular α13, which is believed to play a role in recognition of kinase substrates (12). The two nucleotides of substrate DNA visible in these structures traverse ∼13 Å from the surface of the phosphatase domain to the catalytic center.…”

Section: Resultsmentioning

confidence: 99%

“…Consistent with its role in these DNA repair pathways, PNKP recognizes 3′-phosphate termini in the context of DNA gaps and nicks, as well as doubleand single-strand breaks (11). While recent structural and biochemical approaches have begun to shed light on how the kinase active site recognizes its substrates (12), the way in which substrates are recognized by the distinct phosphatase active site are not well understood. Here we present the results of crystallographic and biochemical studies of PNKP phosphatase and its interactions with DNA substrates.…”

mentioning

confidence: 99%

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

Coquelle

Havali-Shahriari

Bernstein

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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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 ...

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“…This conserved eukaryal protein termed interchangeably PNKP and Pnk1 contains 5'-kinase and 3'-phosphatase domains resembling those of T4 Pnk but arranged in the reverse order, the phosphoesterase domain preceding the kinase domain. The mammalian PNKP is also endowed with an N-terminal FHA (Fork Head Associated) phosphopeptide binding domain that links PNKP to the scaffold proteins XRCC1 and XRCC4 (Bernstein et al, 2009). The latter recruit PNKP to exercise its functions in base excision repair (Hegde et al, 2008) or NHEJ (Lieber, 2008).…”

Section: An Essential Eukaryal Dna Repair Protein Is Related To T4 Pnkmentioning

confidence: 99%

RloC: A Translation-Disabling tRNase Implicated in Phage Exclusion During Recovery from DNA Damage

Kaufmann¹,

Davidov²,

Steinfels-Kohn³

et al. 2011

DNA Repair

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Mechanism of DNA substrate recognition by the mammalian DNA repair enzyme, Polynucleotide Kinase

Cited by 50 publications

References 45 publications

DNA DSB repair pathway choice: an orchestrated handover mechanism

DNA DSB repair pathway choice: an orchestrated handover mechanism

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

RloC: A Translation-Disabling tRNase Implicated in Phage Exclusion During Recovery from DNA Damage

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