Elsevier

DNA Repair

Volume 9, Issue 6, 4 June 2010, Pages 643-652
DNA Repair

RPA physically interacts with the human DNA glycosylase NEIL1 to regulate excision of oxidative DNA base damage in primer-template structures

https://doi.org/10.1016/j.dnarep.2010.02.014Get rights and content

Abstract

The human DNA glycosylase NEIL1, activated during the S-phase, has been shown to excise oxidized base lesions in single-strand DNA substrates. Furthermore, our previous work demonstrating functional interaction of NEIL1 with PCNA and flap endonuclease 1 (FEN1) suggested its involvement in replication-associated repair. Here we show interaction of NEIL1 with replication protein A (RPA), the heterotrimeric single-strand DNA binding protein that is essential for replication and other DNA transactions. The NEIL1 immunocomplex isolated from human cells contains RPA, and its abundance in the complex increases after exposure to oxidative stress. NEIL1 directly interacts with the large subunit of RPA (Kd ∼20 nM) via the common interacting interface (residues 312–349) in NEIL1's disordered C-terminal region. RPA inhibits the base excision activity of both wild-type NEIL1 (389 residues) and its C-terminal deletion CΔ78 mutant (lacking the interaction domain) for repairing 5-hydroxyuracil (5-OHU) in a primer-template structure mimicking the DNA replication fork. This inhibition is reduced when the damage is located near the primer-template junction. Contrarily, RPA moderately stimulates wild-type NEIL1 but not the CΔ78 mutant when 5-OHU is located within the duplex region. While NEIL1 is inhibited by both RPA and Escherichia coli single-strand DNA binding protein, only inhibition by RPA is relieved by PCNA. These results showing modulation of NEIL1's activity on single-stranded DNA substrate by RPA and PCNA support NEIL1's involvement in repairing the replicating genome.

Introduction

Reactive oxygen species (ROS) continuously produced as by-products of cellular respiration and metabolism of toxic compounds, and also generated after inflammation and exposure to ionizing radiation, comprise the most pervasive genotoxic agents [1], [2], [3]. ROS-induced DNA damage includes oxidatively modified bases (i.e., 8-oxoguanine, thymine glycol 5-hydroxyuracil, and formamidopyrimidines, etc.), abasic (apurinic/apyrimidinic; AP) sites and DNA strand breaks. Most of these lesions are potentially mutagenic and have been implicated in the etiology of various diseases including cancer, degenerative neurological disorders, arthritis, and also in aging and cellular toxicity [4], [5], [6], [7], [8], [9]. These base damages are repaired primarily via the DNA base excision repair (BER) pathway, a highly conserved process that is initiated with excision of the lesion by a DNA glycosylase [10].

Four human DNA glycosylases with overlapping and broad substrate range, and belonging to two families named after the Escherichia coli prototype enzymes, are primarily responsible for repairing several dozen oxidatively modified bases. All oxidized base-specific glycosylases possess intrinsic AP lyase activity and cleave the DNA strand at the AP site after base excision [11]. The human Nth family members OGG1 and NTH1 carry out β elimination to produce 3′ phosphodeoxyribose (3′ dRP) terminus at the strand break while the glycosylases in the Nei family possess β,δ-lyase activity to generate 3′ phosphate [12], [13]. We and others have identified and characterized mammalian orthologs of the Nei family which we named NEILs [14], [15], [16], [17], [18]. The 3′dRP or 3′ phosphate blocking group generated by the Nth or Nei type glycosylases is removed in mammalian cells by AP endonuclease (APE1) or polynucleotide kinase (PNK), respectively, to generate 3′ OH [19], [20]. In the basic BER process, DNA polymerase β (Pol β) fills in the single nucleotide gap and in the final step, DNA ligase IIIα (Lig IIIα) seals the nick to restore genome integrity in the single nucleotide (SN) BER pathway [21], [22]. Recent studies in our and other laboratories have shown that the BER pathway is more complex than observed in single nucleotide repair (SN-BER), with cross-talk occurring between the core components of BER and DNA metabolic pathways including transcription and replication. Multiple repair sub-pathways are likely to be active in vivo. Dou et al., demonstrated that NEIL1 and NEIL2 unlike OGG1 and NTH1 are able to excise oxidized base lesions from regions of single-stranded DNA suggesting a role for NEIL-initiated repair of oxidative DNA damage during replication and/or transcription [23].

Our early studies of NEIL1-initiated BER showed pairwise interaction of NEIL1 (and NEIL2) with downstream components of the SN-BER pathway including Pol β, Lig IIIα and XRCC1 suggesting formation of a repair complex in which NEIL1(and NEIL2) play a critical role in coordinating subsequent repair steps after base excision [20], [48]. We have recently shown that NEIL1 also interacts with and is activated by several proteins which are involved in DNA replication or intermediate processing. Thus we observed physical and functional interaction of NEIL1 with Werner syndrome protein (WRN), a RecQ family DNA helicase whose deficiency is associated with early onset of aging and which may restore collapsed replication forks [24]. We also showed binary interaction of NEIL1 with proliferating cell nuclear antigen (PCNA), the sliding clamp responsible for DNA polymerase processivity, and flap endonuclease 1 (FEN1), an essential 5′ endo/exonuclease required for the removal of Okazaki fragments in the lagging strand during DNA replication [25], [26]. All of these proteins stimulate NEIL1 activity with various substrate structures [24], [25], [26]. The replication factor C (RFC), that loads the PCNA clamp onto the primed DNA template, was also identified in the NEIL1 complex [25]. Furthermore, Lu's group, in collaboration with us, showed that NEIL1 stably interacts with and is stimulated by the Rad9–Rad1–Hus1 (9-1-1) complex, a damage-activated DNA sliding clamp that replaces PCNA upon p21 activation and replication arrest [27]. The discovery of functional interaction between NEIL1 and DNA replication-associated proteins, NEIL1's S-phase specific upregulation and activity with single-stranded (ss) DNA substrate prompted us to hypothesize that NEIL1 is involved in preferential repair of oxidative base damage in the replicating genome.

In order to test for physical association of various replication proteins with NEIL1, we carried out systematic Western analysis of the NEIL1 immunocomplex isolated from nuclear extracts of HeLa and HCT 116 cells. We identified replication protein A (RPA), the major ssDNA binding protein in eukaryotes initially discovered as an essential factor in SV40 DNA replication and later shown to be also essential for cellular DNA replication [28], [29], [30]. The heterotrimeric RPA is composed of three subunits namely, 70-kDa RPA1, 32-kDa RPA2, and 14-kDa RPA3 [31]. Although a recent study implicated another mammalian ssDNA binding protein in DNA transactions [32], RPA has been shown to be involved in a variety of DNA damage responses [33]. Native RPA possesses four DNA binding domains (DBD), A–D, which independently bind to ssDNA with decreasing affinity from A to D. The domains (DBD-A, -B and -C) are present in RPAl while DBD-D is located in the RPA2 [34]. The DBDs initiate binding at the 5′ end of ssDNA starting with DBD-A and-B occluding 8–10 nucleotides [35], [36]. Then, via conformational change, DBD-C binds to cover a stretch of 12–23 nucleotides [37]. Finally, co-operative binding of all four DBDs results in a fully extended conformation of RPA covering a stretch of approximately 30 nucleotides [38], [39]. These three distinct states of binding are believed to coexist and native RPA may undergo change between extended and compact conformations depending on the need to cover the ssDNA stretch [40].

RPA (∼105 molecules/cell), one of the more abundant cellular proteins, interacts with a number of proteins in various DNA metabolic pathways including DNA replication, repair, recombination and damage responses [41], [42]. Accumulating evidence indicate that RPA plays an active role in DNA damage responses beyond protection of ssDNA sequences from degradation by nucleases. For example, RPA inhibits APE1's cleavage of the AP site in ssDNA without requiring direct protein–protein interaction [43]. On the other hand, human nuclear uracil-DNA glycosylase (UNG2) individually interacts with PCNA and RPA and colocalizes with these proteins in replication foci. Phosphorylated UNG2 is present in these foci [44]. The S-phase-specific of UNG2-RPA complex was suggested to be involved in post-replicative repair of U misincorporated in nascent DNA [45]. In this study, we show that NEIL1 and RPA are present in a nuclear complex and NEIL1 directly interacts with RPA in vitro. Additionally, RPA inhibits NEIL1's excision of 5-OHU when present in the single-strand segment of a partial duplex oligo resembling primer-template, but it stimulates NEIL1 to repair the lesion when present in the duplex sequence. RPA passively inhibits NEIL1 single-strand lesion excision activity but interacts with the glycosylase for stimulating its double-strand-specific activity, and PCNA is specific for specifically relieving inhibition by RPA but not by E. coli single-strand DNA binding protein (SSB). These results suggest an active role of RPA in controlling NEIL1-dependent repair of oxidative base damage in the replicating genome.

Section snippets

Oligonucleotide substrates

A 51-mer oligo containing 5-OHU at position 26 from the 5′-end or undamaged 51-mer control oligo contained C at position 26 were 32P-labeled at the 5′ terminus with [γ-32P] ATP using T4-PNK prior to annealing when necessary, as described earlier [23]. The sequences in complementary oligos had G opposite the lesion which was used for generating complete or partial duplexes at the 3′ end to produce 3′ primer-template structures as shown in Table 1. To generate the 5′ primer-template structure

In vivo association of NEIL1 with RPA

We recently documented physical and functional interaction of NEIL1 with PCNA, FEN1 and WRN as well as its in vivo association with RFC [24], [25], [26]. Furthermore, we also identified RPA among the proteins present in the NEIL1 immunoprecipitate in our screen for replication-associated proteins (Fig. 1A), suggesting stable association between RPA and NEIL1. We tested the effect of induced oxidative stress caused by the mitochondrial complex III inhibitor antimycin A, on RPA-association in

Discussion

Because of NEIL1's S-phase-dependent upregulation and preference for ssDNA substrates [28], [39], we have proposed its preferential role in repairing lesions in the replicating DNA. Unlike bulky adducts, oxidized bases do not block chain elongation by replicating DNA polymerases δ/ɛ [39], [41], [52]. Unrepaired lesions may be replicated by the replicative or translesion synthesis (TLS) polymerases to generate mutations. We have postulated that free DNA glycosylases or their complexes recruited

Conflict of interest

None.

Acknowledgements

We would like to thank Thomas Wood, Director of the Molecular Genetics Core, Alex Kurosky and Steven Smith of the Biomolecular Resources Facility at UTMB for various services and analysis, and Ms. Wanda Smith for expert secretarial assistance. Research was supported by USPHS grants R01 CA081063 (SM), R01 CA102271 (TKH), P01 CA092584 (SM) and the NIEHS Toxicology Center grant P30 ES06676. CAT was supported by a USPHS predoctoral training grant T32-07254.

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