IN VIVO EXCISION OF PYRIMIDINE DIMERS IS MEDIATED BY A DNA N‐GLYCOSYLASE IN MICROCOCCUS LUTEUS BUT NOT IN HUMAN FIBROBLASTS

Abstract

It has been previously shown that Micrococcus luteus possesses a pyrimidine dimer‐specific endonuclease which in vitro, functions as both an endonuclease and DNA‐glycosylase. To determine if these combined activities function in vivo, we have isolated and examined the excision products of UV‐irradiated M. luteus. In addition, we have devised a procedure to isolate and examine the excision products from UV‐irradiated human fibroblasts to determine if an endonuclease/glycosylase activity functions in the excision of UV‐induced pyrimidine dimers in human fibroblasts. We find that, in vivo, an endonuclease/glycosylase mechanism is utilized extensively in the repair of pyrimidine dimers by M. luteus, but that human fibroblasts do not appear to use this mechanism.

Cancer predisposition, carcinogen hypersensitivity, and aberrant DNA metabolism

“…Our failure to detect free thymine after direct photoreversal of dimer-containing excision products confirms the findings of LaBelle and Linn (1982) that the putative human UV endonuclease does not initiate dimer excision by an N-glycosidic cleavage. Instead, hydrolysis of the intradimer phosphodiester bond appears to constitute the first step in excision repair of dimers in human cells.…”

Human nucleotide excision nuclease removes thymine dimers from DNA by incising the 22nd phosphodiester bond 5' and the 6th phosphodiester bond 3' to the photodimer.

“…Whether only the 29-mer or all three fragments were primary excision products could not be ascertained from this experiment because even a contaminating 25-mer was partially degraded to smaller species (compare lanes 4 and 5). To locate the TOT in the excised fragment and thus more precisely define the 5' 29 incision site, we prepared ccDNA free of the 25-mer contaminant, conducted the excision assay, and then treated the It is known that human cells remove TOTs as oligonucleog; 3, incubated with tides (28) and that nucleotide excision generates repair tolyase and photopatches of 20-30 nt, as determined by density labeling activated DNA was (29)(30)(31), by the bromodeoxyuridine photolysis methods (32) treated with CFE.…”

Mechanisms of DNA Repair by Photolyase and Excision Nuclease (Nobel Lecture)

“…In early work on photolyase, E. coli cells were irradiated with UV in as uspension in abuffer,and then one half was exposed to blue light while the other half was kept in the dark. After these initial findings,r esearch done in numerous labs led to the conclusions summarized in Figure 7: T <>Td imers are removed (excised) from the genome in both E. coli and humans [39,40] in the form of 4to6nucleotide (nt) long oligomers [36,37,[40][41][42] but remain within the cell and are not exported. However,ifthe same experiment was carried out in ab uffer containing glucose as an energy source,i ncubation of UV-irradiated E. coli, in either the dark or the light, resulted in the disappearance of T <>Td imers from the genome.…”