Thermococcus Eurythermalis Endonuclease IV Can Cleave Various Apurinic/Apyrimidinic Site Analogues in ssDNA and dsDNA

Endonuclease IV (EndoIV) is a DNA damage-specific endonuclease that mainly hydrolyzes the phosphodiester bond located at 5′ of an apurinic/apyrimidinic (AP) site in DNA. EndoIV also possesses 3′-exonuclease activity for removing 3′-blocking groups and normal nucleotides. Here, we report that Thermococcus eurythermalis EndoIV (TeuendoIV) shows AP endonuclease and 3′-exonuclease activities. The effect of AP site structures, positions and clustered patterns on the activity was characterized. The AP endonuclease activity of TeuendoIV can incise DNA 5′ to various AP site analogues, including the alkane chain Spacer and polyethylene glycol Spacer. However, the short Spacer C2 strongly inhibits the AP endonuclease activity. The kinetic parameters also support its preference to various AP site analogues. In addition, the efficient cleavage at AP sites requires ≥2 normal nucleotides existing at the 5′-terminus. The 3′-exonuclease activity of TeuendoIV can remove one or more consecutive AP sites at the 3′-terminus. Mutations on the residues for substrate recognition show that binding AP site-containing or complementary strand plays a key role for the hydrolysis of phosphodiester bonds. Our results provide a comprehensive biochemical characterization of the cleavage/removal of AP site analogues and some insight for repairing AP sites in hyperthermophile cells.

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Kinetic Features of 3′-5′ Exonuclease Activity of Human AP-Endonuclease APE1

Molecules ◽

10.3390/molecules23092101 ◽

2018 ◽

Vol 23 (9) ◽

pp. 2101 ◽

Cited By ~ 6

Author(s):

Alexandra Kuznetsova ◽

Olga Fedorova ◽

Nikita Kuznetsov

Keyword(s):

Kinetic Mechanism ◽

Primary Role ◽

Exonuclease Activity ◽

Abasic Site ◽

Ap Endonuclease ◽

Phosphodiester Bond ◽

Short Oligonucleotide ◽

Rate Limiting Step ◽

Ap Sites ◽

The Impact

Human apurinic/apyrimidinic (AP)-endonuclease APE1 is one of the key enzymes taking part in the repair of damage to DNA. The primary role of APE1 is the initiation of the repair of AP-sites by catalyzing the hydrolytic incision of the phosphodiester bond immediately 5′ to the damage. In addition to the AP-endonuclease activity, APE1 possesses 3′-5′ exonuclease activity, which presumably is responsible for cleaning up nonconventional 3′ ends that were generated as a result of DNA damage or as transition intermediates in DNA repair pathways. In this study, the kinetic mechanism of 3′-end nucleotide removal in the 3′-5′ exonuclease process catalyzed by APE1 was investigated under pre-steady-state conditions. DNA substrates were duplexes of deoxyribonucleotides with one 5′ dangling end and it contained a fluorescent 2-aminopurine residue at the 1st, 2nd, 4th, or 6th position from the 3′ end of the short oligonucleotide. The impact of the 3′-end nucleotide, which contained mismatched, undamaged bases or modified bases as well as an abasic site or phosphate group, on the efficiency of 3′-5′ exonuclease activity was determined. Kinetic data revealed that the rate-limiting step of 3′ nucleotide removal by APE1 in the 3′-5′ exonuclease process is the release of the detached nucleotide from the enzyme’s active site.

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Structure of the endonuclease IV homologue fromThermotoga maritimain the presence of active-site divalent metal ions

Acta Crystallographica Section F Structural Biology and Crystallization Communications ◽

10.1107/s1744309110028575 ◽

2010 ◽

Vol 66 (9) ◽

pp. 1003-1012 ◽

Cited By ~ 5

Author(s):

Stephen J. Tomanicek ◽

Ronny C. Hughes ◽

Joseph D. Ng ◽

Leighton Coates

Keyword(s):

Metal Ions ◽

Active Site ◽

Excision Repair ◽

Divalent Metal ◽

Hydroxyl Group ◽

Divalent Metal Ions ◽

Ap Endonuclease ◽

Phosphodiester Bond ◽

Dna Base ◽

Ap Site

The most frequent lesion in DNA is at apurinic/apyrimidinic (AP) sites resulting from DNA-base losses. These AP-site lesions can stall DNA replication and lead to genome instability if left unrepaired. The AP endonucleases are an important class of enzymes that are involved in the repair of AP-site intermediates during damage-general DNA base-excision repair pathways. These enzymes hydrolytically cleave the 5′-phosphodiester bond at an AP site to generate a free 3′-hydroxyl group and a 5′-terminal sugar phosphate using their AP nuclease activity. Specifically,Thermotoga maritimaendonuclease IV is a member of the second conserved AP endonuclease family that includesEscherichia coliendonuclease IV, which is the archetype of the AP endonuclease superfamily. In order to more fully characterize the AP endonuclease family of enzymes, two X-ray crystal structures of theT. maritimaendonuclease IV homologue were determined in the presence of divalent metal ions bound in the active-site region. These structures of theT. maritimaendonuclease IV homologue further revealed the use of the TIM-barrel fold and the trinuclear metal binding site as important highly conserved structural elements that are involved in DNA-binding and AP-site repair processes in the AP endonuclease superfamily.

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Biochemical characterization of recombinant influenza A polymerase heterotrimer complex: Endonuclease activity and evaluation of inhibitors

PLoS ONE ◽

10.1371/journal.pone.0181969 ◽

2017 ◽

Vol 12 (8) ◽

pp. e0181969 ◽

Cited By ~ 1

Author(s):

Weimei Xing ◽

Ona Barauskas ◽

Thorsten Kirschberg ◽

Anita Niedziela-Majka ◽

Michael Clarke ◽

...

Keyword(s):

Influenza A ◽

Biochemical Characterization ◽

Endonuclease Activity

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CiApex1 has AP endonuclease activity and abrogated AP site repair disrupts early embryonic development in Ciona intestinalis

Genes & Genetic Systems ◽

10.1266/ggs.18-00043 ◽

2019 ◽

Vol 94 (2) ◽

pp. 81-93

Author(s):

Kento Igarashi ◽

Masafumi Funakoshi ◽

Seiji Kato ◽

Takahito Moriwaki ◽

Yuichi Kato ◽

...

Keyword(s):

Embryonic Development ◽

Ciona Intestinalis ◽

Early Embryonic Development ◽

Endonuclease Activity ◽

Ap Endonuclease ◽

Ap Site

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Characterization of Abasic Endonuclease Activity of Human Ape1 on Alternative Substrates, as Well as Effects of ATP and Sequence Context on AP Site Incision

Journal of Molecular Biology ◽

10.1016/j.jmb.2008.03.053 ◽

2008 ◽

Vol 379 (1) ◽

pp. 17-27 ◽

Cited By ~ 61

Author(s):

Brian R. Berquist ◽

Daniel R. McNeill ◽

David M. Wilson

Keyword(s):

Endonuclease Activity ◽

Sequence Context ◽

Alternative Substrates ◽

As Effects ◽

Ap Site

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Biochemical Characterization of Metnase’s Endonuclease Activity and Its Role in NHEJ Repair

Biochemistry ◽

10.1021/bi200333k ◽

2011 ◽

Vol 50 (20) ◽

pp. 4360-4370 ◽

Cited By ~ 27

Author(s):

Brian D. Beck ◽

Sung-Sook Lee ◽

Elizabeth Williamson ◽

Robert A. Hromas ◽

Suk-Hee Lee

Keyword(s):

Biochemical Characterization ◽

Endonuclease Activity ◽

Nhej Repair

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Complementary Functions of Plant AP Endonucleases and AP Lyases during DNA Repair of Abasic Sites Arising from C:G Base Pairs

International Journal of Molecular Sciences ◽

10.3390/ijms22168763 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8763

Author(s):

Marina Jordano-Raya ◽

Cristina Beltrán-Melero ◽

M. Dolores Moreno-Recio ◽

M. Isabel Martínez-Macías ◽

Rafael R. Ariza ◽

...

Keyword(s):

Dna Repair ◽

Dna Lesions ◽

Abasic Site ◽

Base Loss ◽

Base Pairs ◽

Ap Endonuclease ◽

Abasic Sites ◽

Ap Sites ◽

Ap Lyase ◽

Ap Site

Abasic (apurinic/apyrimidinic, AP) sites are ubiquitous DNA lesions arising from spontaneous base loss and excision of damaged bases. They may be processed either by AP endonucleases or AP lyases, but the relative roles of these two classes of enzymes are not well understood. We hypothesized that endonucleases and lyases may be differentially influenced by the sequence surrounding the AP site and/or the identity of the orphan base. To test this idea, we analysed the activity of plant and human AP endonucleases and AP lyases on DNA substrates containing an abasic site opposite either G or C in different sequence contexts. AP sites opposite G are common intermediates during the repair of deaminated cytosines, whereas AP sites opposite C frequently arise from oxidized guanines. We found that the major Arabidopsis AP endonuclease (ARP) exhibited a higher efficiency on AP sites opposite G. In contrast, the main plant AP lyase (FPG) showed a greater preference for AP sites opposite C. The major human AP endonuclease (APE1) preferred G as the orphan base, but only in some sequence contexts. We propose that plant AP endonucleases and AP lyases play complementary DNA repair functions on abasic sites arising at C:G pairs, neutralizing the potential mutagenic consequences of C deamination and G oxidation, respectively.

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