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

Byung-In Lee,David M Wilson

doi:10.1074/jbc.274.53.37763

Byung-In Lee, David M Wilson

Open Access

https://doi.org/10.1074/jbc.274.53.37763

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Abstract

The RAD2 family of nucleases includes human XPG (Class I), FEN1 (Class II), and HEX1/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 Mg(2+) being the preferred metal ion. HEX1-N2 is approximately 3-fold less active in Mn(2+)-containing buffers and exhibits <5% activity in the presence of Co(2+), Zn(2+), or Ca(2+). 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.

Highlights

Genomic integrity is maintained by specific cellular responses to genetic damage, such as DNA repair and cell cycle checkpoint controls [1, 2]
RAD2 Class I consists of the XPG1-like proteins that operate in nucleotide excision repair to incise the target strand to the 3Ј-side of the bubble-like, damage-containing structure formed as an intermediate during the repair event [6]
Based on the fact that the yeast proteins exhibit similar instability [37, 38], it seems likely that the C- and N-terminal domains of the EXO1-like proteins exist as distinct entities, separated by a degradationsusceptible connecting region

Summary

Nucleotide sequence

The RAD2 Class III members consist of the EXO1-like enzymes found in yeast, fly, and mammals (20 –26). E. coli polA (the gene encoding PolI) null mutants are inviable when grown on rich medium, a condition thought to promote rapid growth, cell division, and imposing chromosomal replication [29]. These mutants are viable on minimal medium, presumably due to the reduced replication burden. The 5Ј nuclease function of FEN1 is not essential for viability [13, 14, 34, 35], as null mutations in Saccharomyces cerevisiae rad or Schizosaccharomyces pombe rad confer only conditional lethality (i.e. growth observed at 30 °C but not at 37 °C) This finding indicates that an alternative nuclease activity exists at permissive temperatures. Studies presented within indicate that, in addition to RNA primer removal activity [35], the EXO1-like proteins display a FEN1-like flap endonuclease activity, expanding the breadth of their potential functional complementarity and raising new issues regarding the biological synergy of these two RAD2 family members

EXPERIMENTAL PROCEDURES

RESULTS

Specific activity kcata kcat

DISCUSSION