David M. Wilson

The major human abasic endonuclease: formation, consequences and repair of abasic lesions in DNA

DNA continuously suffers the loss of its constituent bases, and thereby, a loss of potentially vital genetic information. Sites of missing bases--termed abasic or apurinic/apyrimidinic (AP) sites--form spontaneously, through damage-induced hydrolytic base release, or by enzyme-catalyzed removal of modified or mismatched bases during base excision repair (BER). In this review, we discuss the structural and biological consequences of abasic lesions in DNA, as well as the multiple repair pathways for such damage, while emphasizing the mechanistic operation of the multi-functional human abasic endonuclease APE1 (or REF-1) and its potential relationship to disease.

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

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.

Second human protein with homology to the Escherichia coli abasic endonuclease exonuclease III

There are two major apurinic/apyrimidinic (AP) endonuclease/3′‐diesterase families designated after the Escherichia coli proteins exonuclease III (ExoIII) and endonuclease IV (EndoIV). These repair proteins function to excise mutagenic and cytotoxic AP sites or 3′‐phosphate/phosphoglycolate groups from DNA. In mammals, the predominant repair endonuclease is Ape1, a homolog of ExoIII, whereas a mammalian homolog to EndoIV has not been identified to date. We have identified a human protein termed Ape2 that represents a subclass of the ExoIII family (exhibiting highest similarity to the Saccharomyces cerevisiae ETH1/APN2 gene product) and maintains many of the essential functional residues of the ExoIII‐like proteins. The human protein is 518 amino acids with a predicted molecular mass of 57.3 kDa and a pI of 8.65. Unlike Ape1, this protein exhibited only weak ability to complement the repair defects of AP endonuclease/3′‐repair‐defective bacteria and yeast. Similarly, a weak, but specific, DNA‐binding and incision activity for abasic site‐containing substrates was observed with partially purified Ape2 protein. APE2 is located on the X chromosome at position p11.21 and consists of six exons. The transcript for APE2 is ubiquitously expressed, suggesting an important function for the encoded protein. An Ape2 green fluorescent fusion protein localized predominantly to the nucleus of HeLa cells, indicating a nuclear function; this localization was dependent on the C‐terminal domain. We discuss our results in the context of the evolutionary conservation of the AP endonuclease families and their divergent activities and biological contributions. Environ. Mol. Mutagen. Environ. Mol. Mutagen. 36:312–324, 2000. Published 2000 Wiley‐Liss, Inc.

Identification of factors interacting with hMSH2 in the fetal liver utilizing the yeast two-hybrid system: in vivo interaction through the C-terminal domains of hEXO1 and hMSH2 and comparative expression analysis

Mutations in DNA mismatch repair (MMR) genes have been shown to segregate with Hereditary Nonpolyposis Colorectal Cancer (HNPCC). However, because many HNPCC families fail to display mutations in known MMR genes, we argued that changes in other components of the MMR pathway may be responsible. The increasing number of proteins reported to interact in the MMR pathway suggests that larger complexes are formed, the composition of which may differ among cell types and tissues. In an attempt to identify tissue-specific MMR-associated factors, we employed the yeast two-hybrid system, using the human hMSH2 as bait and a human fetal liver library as prey. We demonstrate that hMSH2 interacts with a human 5-3 exonuclease 1 (hEXO1/HEX1) and that this interaction is mediated through their C-terminal domains. The hMSH6 protein does not interact with hEXO1 in the two-hybrid system. Dot-blot analysis of multiple tissue RNA revealed that hMSH2 and hEXO1 are coexpressed at high levels in fetal liver as well as in adult testis and thymus. Northern blot analysis also revealed that hEXO1/HEX1 is highly expressed in several liver cancer cell lines as well as in colon and pancreas adenocarcinomas, but not in the corresponding non-neoplastic tissue.