Interaction of the Human DNA Glycosylase NEIL1 with Proliferating Cell Nuclear Antigen

Abstract

NEIL1 and NEIL2 compose a family of DNA glycosylases that is distinct from that of the other two DNA glycosylases, OGG1 and NTH1, all of which are involved in repair of oxidized bases in mammalian genomes. That the NEIL proteins, unlike OGG1 and NTH1, are able to excise base lesions from single-stranded DNA regions suggests their preferential involvement in repair during replication and/or transcription. Previous studies showing S phase-specific activation of NEIL1, but not NEIL2, suggested NEIL1 involvement in the repair of replicating DNA. Here, we show that human NEIL1 stably interacts both in vivo and in vitro with proliferating cell nuclear antigen (PCNA), the sliding clamp for DNA replication. PCNA stimulates NEIL1 activity in excising the oxidized base 5-hydroxyuracil from single-stranded DNA sequences including fork structures. PCNA enhances NEIL1 loading on the substrate. In contrast, although present in the NEIL2 immunocomplex, PCNA does not stimulate NEIL2. NEIL1 interacts with PCNA via a domain that is located in a region near the C terminus, dispensable for base excision activity. The interacting sequence in NEIL1, which lacks the canonical PCNA-binding motif, includes a sequence conserved in DNA polymerase ␦ and implicated in its PCNA binding. Mammalian two-hybrid analysis confirmed PCNA interaction with NEIL1. The G127A mutation in PCNA reduces its stimulatory activity, suggesting that the interdomain connector loop, a common binding interface of PCNA, is involved in NEIL1 binding. These results strongly support in vivo function of NEIL1 in preferential repair of oxidized bases in DNA prior to replication.Reactive oxygen species, ubiquitous genotoxic agents, are continuously generated in vivo as by-products of respiration and are also produced as a result of metabolism of toxic agents or induced during inflammatory responses (1-3). Reactive oxygen species have been implicated in the etiology of a wide variety of diseases ranging from arthritis to cancer and also in aging (4). Reactive oxygen species induce a plethora of oxidatively damaged bases, abasic (apurinic/apyrimidinic (AP)) 5 sites, and strand breaks in the genome that, if left unrepaired, could lead to mutagenesis, apoptosis, senescence, and sporadic cancer (4). Such damage, with the exception of double strand breaks, is repaired primarily via the DNA base excision repair (BER) pathway (5).The BER pathway, first delineated in Escherichia coli, is initiated with excision of the damaged base by a DNA glycosylase. Oxidized base-specific glycosylases, all with intrinsic AP lyase activity, then cleave the DNA strand at the resulting AP site, generating a 3Ј-terminal deoxyribose phosphate or 3Ј-phosphate (6). After removal of this 3Ј-blocking group, the single nucleotide gap is filled in by a DNA polymerase, using the undamaged complementary strand as a template, and DNA ligase finally seals the nick to restore duplex DNA (5, 7). The oxidized base-specific DNA glycosylases have broad substrate specificity commensurate with the large number of s...

Physical and Functional Interactions between Uracil-DNA Glycosylase and Proliferating Cell Nuclear Antigen from the Euryarchaeon Pyrococcus furiosus

“…Consistent with these observations, physical and functional interactions between human UNG2 and PCNA in vitro were confirmed with the purified proteins (16). Besides UNG2, several DNA glycosylases reportedly interact with PCNA (17)(18)(19). It is widely known that many other replicative and repair enzymes interact with PCNA, via a consensus sequence called the PCNA-interacting protein (PIP) box (15).…”

Cockayne Syndrome Group B Protein Stimulates Repair of Formamidopyrimidines by NEIL1 DNA Glycosylase

“…2D). Dou et al (45) recently showed that proliferating cell nuclear antigen also interacts with and stimulates NEIL1 incision activity. However, in that study proliferating cell nuclear antigen stimulated NEIL1 incision activity up to 3-fold at a much higher molar excess of proliferating cell nuclear antigen (ϳ1:83.3).…”

RNA editing changes the lesion specificity for the DNA repair enzyme NEIL1

“…S2) and others have described overexpression of the edited form (R242) in mammalian cells (30,33). In addition, several studies using edited NEIL1 have shown that removal of 5-OHU or FapyG by NEIL1 is enhanced by the presence of protein-binding partners (e.g., PCNA, FEN1, and CSB) (33)(34)(35). The stimulatory affects afforded by protein partners may depend on the form of NEIL1 used as well as the nature of the lesion and its nucleic acid context.…”