Specificity of the dRP/AP Lyase of Ku Promotes Nonhomologous End Joining (NHEJ) Fidelity at Damaged Ends

Strande, Natasha T.; Roberts, Steven A.; Oh, Sehyun; Hendrickson, Eric A.; Ramsden, Dale A.

doi:10.1074/jbc.m111.329730

Cited by 47 publications

(59 citation statements)

References 32 publications

(40 reference statements)

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“…The AP sites near the 5′ end, which are not deeply embedded into duplex part of DNA, were considered to be relevant for direct NHEJ. Ku did not possess the AP lyase activity when an AP site was embedded in the duplex part of DNA for more than 3 bp [25]. This observation is in agreement with our results [13].…”

Section: Ku Interaction With Ap Sitessupporting

confidence: 93%

“…At the same time, it has been found that the lysine(s) of Ku80 can substitute for the mutated lysines of Ku70 with only partial decrease in the Ku activity on the AP site in the 5′ overhang [24,25].…”

Section: Ku Interaction With Ap Sitesmentioning

confidence: 98%

“…Both these activities are necessary for removing the groups, which block DNA repair during the preparation of DNA ends to ligation in NHEJ. More comprehensive analysis of substrate specificity of Ku's 5′-dRP/AP lyase revealed that Ku has drastically weaker AP lyase activity in the 3′ overhang context as compared to the 5′ overhang substrates [25]. The idea is the following.…”

Section: Ku Interaction With Ap Sitesmentioning

confidence: 99%

“…Ku did not possess the AP lyase activity when an AP site was embedded in the duplex part of DNA for more than 3 bp [25]. This observation is in agreement with our results [13].Interestingly, the authors considered the lysines in the N-terminal part of Ku70 as the main nucleophiles responsible for AP site cleavage; no cross-links with Ku70 were found for AP DNA with the AP site located within DS part of DNA that correlates with undetectable cleavage of AP sites [25]. At the same time, it has been found that the lysine(s) of Ku80 can substitute for the mutated lysines of Ku70 with only partial decrease in the Ku activity on the AP site in the 5′ overhang [24,25].…”

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confidence: 96%

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Ku Antigen Interaction with Apurinic/Apyrimidinic Sites: Nonhomologous End Joining Vs Base Excision Repair

Kosova¹,

Lavrik²,

Ходырева³

2014

MOJPB

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IntroductionThe apurinic/apyrimidinic (AP) site is considered to be a common lesion in genomic DNA, arising at a frequency of 10,000 to 50,000 lesions per mammalian cell per day [1]. The number of AP sites can increase dramatically under stressful conditions such as X-ray or UV light irradiation and alkylating agent exposure. If unrepaired, AP sites present mutagenic and cytotoxic consequences to the cell [2]. The loss of DNA bases and attendant formation of AP sites in DNA occurs spontaneously as a result of hydrolytic cleavage of N-glycosylic bonds or through glycosylasecatalyzed removal of damaged bases during the early stage of base excision repair (BER) [3]. The steady-state levels of AP sites in mammalian cells are in the range of approximately 1 site per 10 6 nucleotides [4]. Repair of AP sites is thought to be extremely rapid [5]. At the same time, it has been found that a part of AP sites can persist in genomic DNA for a rather long period [6]. This observation raised a question about nature of these slowly repaired AP sites and the mechanisms of prompt and dilatory repair. It is widely accepted that, irrespectively of their origin, AP sites are further processed by the BER machinery. In most cases, the repair of AP sites is initiated by hydrolytic cleavage 5′ to an AP site catalyzed by apurinic/apyrimidinic endonuclease 1 (APE1) [7]. AP sites also can be incised through β-elimination Ku Antigen Interaction with Apurinic/Apyrimidinic Sites: Nonhomologous End Joining Vs Base Excision Repair via the activity of DNA glycosylases and other enzymes with associated AP lyase activity [3,7]. In this case, strand incision occurs 3′ to an AP site, generating α,β-unsaturated aldehyde (α,β-4-hydroxypenten-2-al) at the 3′-margin and phosphate at the 5′-margin, and this intermediate is further processed by the downstream BER enzymes [7]. This scenario appears to be realized in the case of isolated AP sites, which are generated under moderate level of DNA damage. But under some highly stressful conditions, e.g. exposure to ionizing radiation, AP sites can be a part of multiply damaged sites containing also oxidized bases, and single-or double-strand breaks (SSBs or DSBs) [8,9]. Indeed, among additional types of damage produced by high linear energy transfer irradiation, AP sites, within 8-10 bp of a DS end, were found to be most frequent [10]. the following steps. First, the preparative BHT of cell extract proteins with 32 P-labelled AP DNA, which contains a biotin moiety, is performed. Second, the cross-linked protein is purified by affinity chromatography on streptavidin-coated paramagnetic beads and resolved by SDS-PAGE. A well-defined Coomassie stained protein band precisely corresponding to the radioactive label is excised from the gel, and the protein is subjected to ingel trypsin digestion and MALDI-TOF-MS. Results from the MS analyses are searched against a database to identify the crosslinked protein. It should be pointed out that in this approach AP sites play two roles, the naturally occurring DNA lesion an...

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Section: Ku Interaction With Ap Sitessupporting

confidence: 93%

Section: Ku Interaction With Ap Sitesmentioning

confidence: 98%

Section: Ku Interaction With Ap Sitesmentioning

confidence: 99%

mentioning

confidence: 96%

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Ku Antigen Interaction with Apurinic/Apyrimidinic Sites: Nonhomologous End Joining Vs Base Excision Repair

Kosova¹,

Lavrik²,

Ходырева³

2014

MOJPB

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“…Even Pol4 activity, recruited to double-strand break ends by NHEJ core factors, is dispensable for 5 0 -dRP removal during joining in Saccharomyces cerevisiae [Daley and Wilson, 2008]. Our group's study indicates that this function is replaced during NHEJ by a 5 0 -dRP/AP lyase activity resident in the NHEJ factor Ku [Roberts et al, 2010], and which has a substrate specificity reciprocal to Pol X members; Ku is active in excising abasic sites only when they are within 4 bp of a double-strand break terminus [Strande et al, 2012].…”

Section: Catalytic Domainmentioning

confidence: 85%

DNA polymerases in nonhomologous end joining: Are there any benefits to standing out from the crowd?

Ramsden

Asagoshi

2012

Environ and Mol Mutagen

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Chromosome breaks, often with damaged or missing DNA flanking the break site, are an important threat to genome stability. They are repaired in vertebrates primarily by nonhomologous end joining (NHEJ). NHEJ is unique among the major DNA repair pathways in that a continuous template cannot be used by DNA polymerases to instruct replacement of damaged or lost DNA. Nevertheless, at least 3 out of the 17 mammalian DNA polymerases are specifically employed by NHEJ. Biochemical and structural studies are further revealing how each of the polymerases employed by NHEJ possesses distinct and sophisticated means to overcome the barriers this pathway presents to polymerase activity. Still unclear, though, is how the resulting network of overlapping and nonoverlapping polymerase activities contributes to repair in cells. Environ. Mol. Mutagen. 53:741-751, 2012. V V C 2012 Wiley Periodicals, Inc.

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Recognition and repair of chemically heterogeneous structures at DNA ends

Andres

Schellenberg

Wallace

et al. 2014

Environ and Mol Mutagen

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Exposure to environmental toxicants and stressors, radiation, pharmaceutical drugs, inflammation, cellular respiration, and routine DNA metabolism all lead to the production of cytotoxic DNA strand breaks. Akin to splintered wood, DNA breaks are not “clean”. Rather, DNA breaks typically lack DNA 5'-phosphate and 3'-hydroxyl moieties required for DNA synthesis and DNA ligation. Failure to resolve damage at DNA ends can lead to abnormal DNA replication and repair, and is associated with genomic instability, mutagenesis, neurological disease, ageing and carcinogenesis. An array of chemically heterogeneous DNA termini arises from spontaneously generated DNA single-strand and double-strand breaks (SSBs and DSBs), and also from normal and/or inappropriate DNA metabolism by DNA polymerases, DNA ligases and topoisomerases. As a front line of defense to these genotoxic insults, eukaryotic cells have accrued an arsenal of enzymatic first responders that bind and protect damaged DNA termini, and enzymatically tailor DNA ends for DNA repair synthesis and ligation. These nucleic acid transactions employ direct damage reversal enzymes including Aprataxin (APTX), Polynucleotide kinase phosphatase (PNK), the tyrosyl DNA phosphodiesterases (TDP1 and TDP2), the Ku70/80 complex and DNA polymerase β (POLβ). Nucleolytic processing enzymes such as the MRE11/RAD50/NBS1/CtIP complex, Flap endonuclease (FEN1) and the apurinic endonucleases (APE1 and APE2) also act in the chemical "cleansing" of DNA breaks to prevent genomic instability and disease, and promote progression of DNA- and RNA-DNA damage response (DDR and RDDR) pathways. Here, we provide an overview of cellular first responders dedicated to the detection and repair of abnormal DNA termini.

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Specificity of the dRP/AP Lyase of Ku Promotes Nonhomologous End Joining (NHEJ) Fidelity at Damaged Ends

Cited by 47 publications

References 32 publications

Ku Antigen Interaction with Apurinic/Apyrimidinic Sites: Nonhomologous End Joining Vs Base Excision Repair

Ku Antigen Interaction with Apurinic/Apyrimidinic Sites: Nonhomologous End Joining Vs Base Excision Repair

DNA polymerases in nonhomologous end joining: Are there any benefits to standing out from the crowd?

Recognition and repair of chemically heterogeneous structures at DNA ends

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