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The biochemical role of the human NEIL1 and NEIL3 DNA glycosylases on model DNA replication forks

The biochemical role of the human NEIL1 and NEIL3 DNA glycosylases on model DNA replication forks
The biochemical role of the human NEIL1 and NEIL3 DNA glycosylases on model DNA replication forks

Endonuclease VIII-like (NEIL) 1 and 3 proteins eliminate oxidative DNA base damage and psoralen DNA interstrand crosslinks through initiation of base excision repair. Current evidence points to a DNA replication associated repair function of NEIL1 and NEIL3, correlating with induced expression of the proteins in S/G2 phases of the cell cycle. However previous attempts to express and purify recombinant human NEIL3 in an active form have been challenging. In this study, both human NEIL1 and NEIL3 have been expressed and purified from E. coli, and the DNA glycosylase activity of these two proteins confirmed using single-and double-stranded DNA oligonucleotide substrates containing the oxidative bases, 5-hydroxyuracil, 8-oxoguanine and thymine glycol. To determine the biochemical role that NEIL1 and NEIL3 play during DNA replication, model replication fork substrates were designed containing the oxidized bases at one of three specific sites relative to the fork. Results indicate that whilst specificity for 5-hydroxyuracil and thymine glycol was observed, NEIL1 acts preferentially on double-stranded DNA, including the damage upstream to the replication fork, whereas NEIL3 preferentially excises oxidized bases from single stranded DNA and within open fork structures. Thus, NEIL1 and NEIL3 act in concert to remove oxidized bases from the replication fork.

Base excision repair, DNA damage, DNA repair, DNA replication, NEI-like DNA glycosylases
2073-4425
1-11
Albelazi, Mustafa S.
dfe9a834-59cd-48ea-8b9f-d319cc7ef7c6
Martin, Peter R.
ecb906d6-5286-4734-841b-a168f9fd7cc2
Mohammed, Soran
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Mutti, Luciano
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Parsons, Jason L.
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Elder, Rhoderick H.
b5bb730a-c05b-4b14-93d4-b4bdb549a7a0
Albelazi, Mustafa S.
dfe9a834-59cd-48ea-8b9f-d319cc7ef7c6
Martin, Peter R.
ecb906d6-5286-4734-841b-a168f9fd7cc2
Mohammed, Soran
ba6e34b4-419f-4b3d-b50a-2abdf8ce50c5
Mutti, Luciano
eba324c3-c849-43dd-b0d7-2e26ce294543
Parsons, Jason L.
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Elder, Rhoderick H.
b5bb730a-c05b-4b14-93d4-b4bdb549a7a0

Albelazi, Mustafa S., Martin, Peter R., Mohammed, Soran, Mutti, Luciano, Parsons, Jason L. and Elder, Rhoderick H. (2019) The biochemical role of the human NEIL1 and NEIL3 DNA glycosylases on model DNA replication forks. Genes, 10 (4), 1-11, [315]. (doi:10.3390/genes10040315).

Record type: Article

Abstract

Endonuclease VIII-like (NEIL) 1 and 3 proteins eliminate oxidative DNA base damage and psoralen DNA interstrand crosslinks through initiation of base excision repair. Current evidence points to a DNA replication associated repair function of NEIL1 and NEIL3, correlating with induced expression of the proteins in S/G2 phases of the cell cycle. However previous attempts to express and purify recombinant human NEIL3 in an active form have been challenging. In this study, both human NEIL1 and NEIL3 have been expressed and purified from E. coli, and the DNA glycosylase activity of these two proteins confirmed using single-and double-stranded DNA oligonucleotide substrates containing the oxidative bases, 5-hydroxyuracil, 8-oxoguanine and thymine glycol. To determine the biochemical role that NEIL1 and NEIL3 play during DNA replication, model replication fork substrates were designed containing the oxidized bases at one of three specific sites relative to the fork. Results indicate that whilst specificity for 5-hydroxyuracil and thymine glycol was observed, NEIL1 acts preferentially on double-stranded DNA, including the damage upstream to the replication fork, whereas NEIL3 preferentially excises oxidized bases from single stranded DNA and within open fork structures. Thus, NEIL1 and NEIL3 act in concert to remove oxidized bases from the replication fork.

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genes-10-00315-v2 - Version of Record
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More information

Accepted/In Press date: 17 April 2019
e-pub ahead of print date: 23 April 2019
Keywords: Base excision repair, DNA damage, DNA repair, DNA replication, NEI-like DNA glycosylases

Identifiers

Local EPrints ID: 432473
URI: http://eprints.soton.ac.uk/id/eprint/432473
ISSN: 2073-4425
PURE UUID: 770165b3-e781-49c6-b5ec-200365cdc3b2

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Date deposited: 17 Jul 2019 16:30
Last modified: 07 Oct 2020 00:38

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