Interaction of the human DNA glycosylase NEIL1 with proliferating cell nuclear antigen. The potential for replication-associated repair of oxidized bases in mammalian genomes.
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 delta 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.
Dou
Hong
H
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-1079, University of Texas Medical Branch, Galveston, Texas 77555-1079.
Theriot
Corey A
CA
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-1079, University of Texas Medical Branch, Galveston, Texas 77555-1079.
Das
Aditi
A
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-1079, University of Texas Medical Branch, Galveston, Texas 77555-1079.
Hegde
Muralidhar L
ML
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-1079, University of Texas Medical Branch, Galveston, Texas 77555-1079.
Matsumoto
Yoshihiro
Y
Department of Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Boldogh
Istvan
I
Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas 77555-1079.
Hazra
Tapas K
TK
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-1079, University of Texas Medical Branch, Galveston, Texas 77555-1079.
Bhakat
Kishor K
KK
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-1079, University of Texas Medical Branch, Galveston, Texas 77555-1079.
Mitra
Sankar
S
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-1079, University of Texas Medical Branch, Galveston, Texas 77555-1079. Electronic address: samitra@utmb.edu.
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D045647:DNA Glycosylases / Q000737:chemistry / Q000378:metabolism*; D005786:Gene Expression Regulation; D016678:Genome; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010100:Oxygen / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D011485:Protein Binding; D016196:S Phase; D020798:Two-Hybrid System Techniques
D018809:Proliferating Cell Nuclear Antigen; D004247:DNA; D045647:DNA Glycosylases; C473634:NEIL1 protein, human; D010100:Oxygen