NDLI: The RAD2 Domain of Human Exonuclease 1 Exhibits 5′ to 3′ Exonuclease and Flap Structure-specific Endonuclease Activities

Content Provider	Journal of Biological Chemistry (JBC)
Author	Lee, Byung-In Iii, David M. Wilson Wilson, David M.
Abstract	The RAD2 family of nucleases includes humanXPG (Class I), FEN1 (Class II), andHEX1/hEXO1 (Class III) products gene. These proteins exhibit a blend of substrate specific exo- and endonuclease activities and contribute to repair, recombination, and/or replication. To date, the substrate preferences of the EXO1-like Class III proteins have not been thoroughly defined. We report here that the RAD2 domain of human exonuclease 1 (HEX1-N2) exhibits both a robust 5′ to 3′ exonuclease activity on single- and double-stranded DNA substrates as well as a flap structure-specific endonuclease activity but does not show specific endonuclease activity at 10-base pair bubble-like structures, G:T mismatches, or uracil residues. Both the 5′ to 3′ exonuclease and flap endonuclease activities require a divalent metal cofactor, with Mg2+ being the preferred metal ion. HEX1-N2 is ∼3-fold less active in Mn2+-containing buffers and exhibits <5% activity in the presence of Co2+, Zn2+, or Ca2+. The optimal pH range for the nuclease activities of HEX1-N2 is 7.2–8.2. The specific activity of its 5′ to 3′ exonuclease function is 2.5–7-fold higher on blunt end and 5′-recessed double-stranded DNA substrates compared with duplex 5′-overhang or single-stranded DNAs. The flap endonuclease activity of HEX1-N2 is similar to that of human flap endonuclease-1, both in terms of turnover efficiency (k cat) and site of incision, and is as efficient (k cat/K m) as its exonuclease function. The nuclease activities of HEX1-N2 described here indicate functions for the EXO1-like proteins in replication, repair, and/or recombination that may overlap with human flap endonuclease-1.
Related Links	http://www.jbc.org/content/274/53/37763.abstract
Ending Page	37769
Starting Page	37763
Page Count	7
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	53
Volume Number	274
DOI	10.1074/jbc.274.53.37763
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	1999-12-31
Access Restriction	Open
Subject Keyword	Xeroderma pigmentosum group G (XPG) Xeroderma pigmentosum group F (XPF) Double-stranded (ds) Dithiothreitol (DTT) Flap endonuclease-1 (FEN1) Human exonuclease 1 (HEX1/hEXO1) MutS homolog 2 (MSH2) Nucleotide (nt) Proliferating cell nuclear antigen (PCNA) Single-stranded (ss) Isopropyl-d-thiogalactopyranoside (IPTG) Polymerase chain reaction (PCR) 4-morpholinepropanesulfonic acid (MOPS) 4-morpholineethanesulfonic acid (MES) 3-[cyclohexylamino]-2-hydroxy-1-propanesulfonic acid (CAPSO) 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid (Taps) Specific activity (S.A.) Human (h) NUCLEIC ACIDS, PROTEIN SYNTHESIS, AND MOLECULAR GENETICS
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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