XRCC1 Stimulates Polynucleotide Kinase by Enhancing Its Damage Discrimination and Displacement from DNA Repair Intermediates

Mani, Rajam S.; Fanta, Mesfin; Karimi‐Busheri, Feridoun; Silver, Elizabeth T.; Virgen, César A.; Caldecott, Keith W.; Cass, Carol E.; Weinfeld, Michael

doi:10.1074/jbc.m704867200

Cited by 46 publications

(62 citation statements)

References 41 publications

(64 reference statements)

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“…One possibility is that interaction with XRCC1 ensures that PNK activity is not rate limiting during SSBR in vivo. This idea is consistent with the observation that XRCC1 stimulates PNK activity in vitro at limiting concentrations of the latter enzyme, possibly by dissociating PNK from its DNA product and thereby increasing the enzyme turnover rate (32,54). However, interaction with XRCC1 also might promote the recruitment or accumulation of PNK at chromosomal SSBs, because the appearance of this protein in subnuclear foci is reduced in EM9-XH CKM cells following oxidative stress (30).…”

Section: Discussionsupporting

confidence: 77%

DNA 3′-Phosphatase Activity Is Critical for Rapid Global Rates of Single-Strand Break Repair following Oxidative Stress

Breslin

Caldecott

2009

Molecular and Cellular Biology

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Oxidative stress is a major source of chromosome single-strand breaks (SSBs), and the repair of these lesions is retarded in neurodegenerative disease. The rate of the repair of oxidative SSBs is accelerated by XRCC1, a scaffold protein that is essential for embryonic viability and that interacts with multiple DNA repair proteins. However, the relative importance of the interactions mediated by XRCC1 during oxidative stress in vivo is unknown. We show that mutations that disrupt the XRCC1 interaction with DNA polymerase ␤ or DNA ligase III fail to slow SSB repair in proliferating CHO cells following oxidative stress. In contrast, mutation of the domain that interacts with polynucleotide kinase/phosphatase (PNK) and Aprataxin retards repair, and truncated XRCC1 encoding this domain fully supports this process. Importantly, the impact of mutating the protein domain in XRCC1 that binds these end-processing factors is circumvented by the overexpression of wild-type PNK but not by the overexpression of PNK harboring a mutated DNA 3-phosphatase domain. These data suggest that DNA 3-phosphatase activity is critical for rapid rates of chromosomal SSB repair following oxidative stress, and that the XRCC1-PNK interaction ensures that this activity is not rate limiting in vivo.Oxidative stress can have a major influence on genome integrity and cell survival and is an etiological factor in a number of neurological human diseases. Of these, several are associated with defects in the repair of DNA damage, including xeroderma pigmentosum (XP), ataxia telangiecatsia (A-T), ataxia oculomotor apraxia 1 (AOA1), and spinocerebellar ataxia with axonal neuropathy 1 (SCAN1) (1,28,34,37). The neuropathology evident in XP most likely reflects an inability to repair one or more single-strand oxidative adducts by nucleotide excision repair. In contrast, A-T is associated with cellular defects in the repair of DNA double-strand breaks (DSBs) and AOA1 and SCAN1 with defects in the repair of DNA single-strand breaks (SSBs). SSBs are the commonest DNA lesions arising in cells, and if they are not rapidly repaired they can inhibit transcription and/or generate replication-associated DSBs (3,26,59,60). The repair of oxidative SSBs involves DNA damage detection by PARP-1 followed by recruitment of the enzymes required for subsequent steps of the repair process, which include DNA end processing, DNA gap filling, and DNA ligation (9, 19). Many of the enzymes implicated in these steps interact physically with XRCC1, including DNA polynucleotide kinase (PNK) (54), Aprataxin (APTX) (13,14,18,31,44), DNA polymerase ␤ (Pol ␤) (10, 27), and DNA ligase III␣ (Lig3␣) (11,12). This has prompted the hypothesis that XRCC1 is a scaffold protein that recruits, stabilizes, and/or stimulates SSB repair (SSBR) enzymes at chromosomal SSBs, thereby accelerating the overall process (8, 9). While in vitro analyses generally are consistent with this idea, including the observation that XRCC1 mutation (50, 58), deletion (49), or depletion (6) retards the rate of chromos...

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Section: Discussionsupporting

confidence: 77%

DNA 3′-Phosphatase Activity Is Critical for Rapid Global Rates of Single-Strand Break Repair following Oxidative Stress

Breslin

Caldecott

2009

Molecular and Cellular Biology

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“…Consistent with this model, PNKP has been shown to have significant affinity for the products of both its kinase and phosphatase domains (22). Interactions with the single-strand repair scaffold protein, XRCC1 or the NHEJ scaffold protein, XRCC4, facilitate PNKP product release, consistent with the idea that these scaffold proteins organize multistep, coordinated repair of DNA damage (23)(24)(25).…”

Section: Resultssupporting

confidence: 58%

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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“…Thus, it is likely that the FHA domain of PNKP recognizes phosphate groups in other molecule besides PAR, which is important for the stability of PNKP at DNA damage sites. It has been reported that PNKP interacted with XRCC1 (Whitehouse et al 2001;Mani et al 2007), which also recognizes PAR at DNA damage sites. However, the recruitment of PNKP to DNA damage sites is independent of XRCC1 (Supplemental Fig.…”

Section: Computational Analysis Of the Par-binding Pockets In The Fhamentioning

confidence: 99%

The FHA and BRCT domains recognize ADP-ribosylation during DNA damage response

et al. 2013

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Poly-ADP-ribosylation is a unique post-translational modification participating in many biological processes, such as DNA damage response. Here, we demonstrate that a set of Forkhead-associated (FHA) and BRCA1 C-terminal (BRCT) domains recognizes poly(ADP-ribose) (PAR) both in vitro and in vivo. Among these FHA and BRCT domains, the FHA domains of APTX and PNKP interact with iso-ADP-ribose, the linkage of PAR, whereas the BRCT domains of Ligase4, XRCC1, and NBS1 recognize ADP-ribose, the basic unit of PAR. The interactions between PAR and the FHA or BRCT domains mediate the relocation of these domain-containing proteins to DNA damage sites and facilitate the DNA damage response. Moreover, the interaction between PAR and the NBS1 BRCT domain is important for the early activation of ATM during DNA damage response and ATM-dependent cell cycle checkpoint activation. Taken together, our results demonstrate two novel PAR-binding modules that play important roles in DNA damage response.

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XRCC1 Stimulates Polynucleotide Kinase by Enhancing Its Damage Discrimination and Displacement from DNA Repair Intermediates

Cited by 46 publications

References 41 publications

DNA 3′-Phosphatase Activity Is Critical for Rapid Global Rates of Single-Strand Break Repair following Oxidative Stress

DNA 3′-Phosphatase Activity Is Critical for Rapid Global Rates of Single-Strand Break Repair following Oxidative Stress

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

The FHA and BRCT domains recognize ADP-ribosylation during DNA damage response

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