Excision of Pyrimidine Dimers from Ultraviolet‐Irradiated DNA by Exonucleases from Mammalian Cells

Abstract

Two DNA specific exonucleases, DNase I11 and DNase IV, are present in mammalian cell nuclei. The activity of the purified enzymes with ultraviolet-irradiated polydeoxynucleotides and DNA as substrates has been investigated. DNase I11 shows a very poor ability to release pyrimidine dimers, although excision can be demonstrated by using high concentrations of enzyme. In contrast, DNase IV liberates h e r s in an effective fashion in vitro, and is a likely candidate to serve in this function also in vivo. The pyrimidine dimers are released by DNase IV as parts of oligonucleotides containing 5-8 residues. Neither of the two exonucleases is markedly inhibited by caffeine.When living cells are exposed to ultraviolet light, the main lesions produced are pyrimidine dimers in DNA, in particular dimers between adjacent thymine residues [l]. Such dimers are formed with similar efficiency in bacterial and mammalian cells [2], and both types of cells possess repair mechanisms that reduce the damaging effect of these structural defects in the DNA. One mechanism of repair depends on the excision of the pyrimidine dimers, followed by repair replication a t the single-stranded gaps thus produced in the DNA, and subsequent rejoining of the strand interruptions [3-71. While this type of DNA repair was originally discovered and characterized in microbial systems, the processes of pyrimidine dimer excision, repair replication, and joining of broken strands have now also been demonstrated to occur in mammalian cells as a consequence of radiation damage [8-lo]. Moreover, this mode of DNA repair is apparently of physiological sigdcance in man, as a loss of the ability to remove pyrimidine dimers from DNA seems to be the cause of the hereditary disease Xeroderma pigmentosum in which the patients are abnormally sensitive to ultraviolet light [ 111.It has been shown in bacteria that the excision of pyrimidine dimers from DNA requires two different enzymes : an endonuclease that specifically attacks ultraviolet-irradiated DNA and catalyzes the formaUnusual Abbreviations. Poly(dA), a homopolymer of deoxyadenylate ; poly(dT), a homopolymer of deoxythymidylate; poly(dA) * poly(dT), a double-stranded polymer composed of poly(dA) and poly(dT) ; poly[d(A-T)], an alternating double-stranded copolymer of deoxyadenylate and deoxythymidylate.All tritium-labeled polymers are labeled in the thymine residues.tion of a single-strand break close to the dimer, and an exonuclease that attacks at the strand interruption and has the property of being able to effectively release pyrimidine dimers from DNA [12,13]. The situation in mammalian cells again seems similar, as cells from a case of Xeroderma pigmentosum could excise damaged residues from irradiated DNA if adjacent single-strand breaks were introduced by non-enzymatic artificial means, i. e . the required endonuclease activity apparently was lacking but the exonuclease was present [la].Two different DNA-specific exonucleases, localized in the cell nuclei, have recently been found in mammalian tissues....

The rad18 Gene of Schizosaccharomyces pombe Defines a New Subgroup of the SMC Superfamily Involved in DNA Repair

“…The mammalian highly conserved homolog of rad2 (16, 31) encodes a structurespecific endonuclease, called FEN-1, whose preferred substrate is a ''flap'' structure (15,16). This protein is also identical to the enzyme DNase IV (38), first isolated by Lindahl (25). Incision of irradiated DNA by the UV endonuclease could provide a substrate for this enzyme to remove a damage-containing DNA fragment.…”

Increased expression and no mutation of the Flap endonuclease (FEN1) gene in human lung cancer

“…Further, the tumors from these mice showed MSI, suggesting that decreased expression of Fen1 may be attributable to MSI of these cancer cells. FEN1 is responsible for DNA replication and base excision repair (BER) pathways and has been shown to play an important role in the integrity of genome (Lindahl, 1971;Guggenheimer et al, 1984;Siegal et al, 1992;Lieber, 1994, 1995;Kim et al, 1998). In addition, there is an evidence that alteration of the BER system occurs in human lung cancer.…”

Haploinsufficiency of Flap endonuclease ( Fen1 ) leads to rapid tumor progression

“…FEN1 functions in the processing of the 5Ј ends of Okazaki fragments in lagging strand synthesis and long patch-base excision type repair (32)(33)(34)(35)(36)(37). FEN1 protein binds to proliferating nuclear cell antigen (38)(39) and potentially competes with p21, xeroderma pigmentosum gene product, 5Ј methyl cytosine methyl transferase, and other proteins for a specific binding motif, implying roles for FEN1 in DNA replication, repair, epigenetic inheritance, and cell cycle control.…”