Purification of Escherichia coli DNA photolyase.

Escherichia coli photolyase is a DNA repair enzyme which monomerizes pyrimidine dimers, the major UV photoproducts in DNA, to pyrimidines in a light-dependent reaction. We recently described the construction of a tac-phr plasmid that greatly overproduces the enzyme (Sancar, G. B., Smith, F. W., and Sancar, A. (1983) Nucleic Acids Res. 11, 6667-6678). Using a strain carrying the overproducing plasmid as the starting material, we have developed a purification procedure that yields several milligrams of apparently homogeneous enzyme. The purified protein is a single polypeptide that has an apparent Mr of 49,000 under both denaturing and nondenaturing conditions. The enzyme has no requirement for divalent cations and it restores the biological activity of irradiated DNA only in the presence of photoreactivating light. The purified photolyase has a turnover number of 2.4 dimers/molecule/min; this value agrees well with the in vivo rate of photoreactivation in E. coli

Transcription preferentially inhibits nucleotide excision repair of the template DNA strand in vitro

It has been reported that pyrimidine dimers (pyrimidine mean value of pyrimidine) are removed preferentially from actively transcribing genes. Furthermore, the preferential repair is restricted to the transcribed strand of these genes. Currently there is no mechanistic explanation for these phenomena. In this study we investigated the effect of transcription on nucleotide excision repair using defined Escherichia coli systems consisting of DNA substrates containing a strong promoter and either (a) a T mean value of T at a defined position in the nontranscribed or transcribed strand or (b) photoproducts randomly distributed in both strands, as well as transcription and nucleotide excision repair enzymes. While a T mean value of T in the nontranscribed strand had no effect on transcription, a photodimer in the transcribed strand blocked transcription causing RNA polymerase to stall at the T mean value of T site. This stalled elongation complex inhibited the excision of the photodimer by (A)BC excinuclease resulting in a net effect of preferential repair of the nontranscribed strand in a mixture containing both substrates. Similarly, when we conducted transcription/repair experiments with a superhelical plasmid no enhanced repair of the transcribed gene was observed compared to nontranscribed regions. We conclude that RNA polymerase stalled at a photodimer does not direct the (A)BC excinuclease to the damaged template strand and therefore cannot account for the strand-specific repair observed in vivo

Cooperative activation of the ATR checkpoint kinase by TopBP1 and damaged DNA

TopBP1, acting in concert with DNA containing bulky base lesions, stimulates ATR kinase activity under physiologically relevant reaction conditions. Here, we analyze the roles of the three components in ATR activation: DNA, base damage and TopBP1. We show that base adducts caused by a potent carcinogen, benzo[a]pyrene diol epoxide (BPDE), constitute a strong signal for TopBP1-dependent ATR kinase activity on Chk1 and p53. We find that the C-terminus of TopBP1 binds preferentially to damaged DNA and is sufficient to mediate damaged DNA-dependent ATR activation in a manner similar to full-length TopBP1. Significantly, we find that stimulation of ATR by BPDE-damaged DNA exhibits strong dependence on the length of DNA, with essentially no stimulation with fragments of 0.2 kb and reaching maximum stimulation with 2 kb fragments. Moreover, TopBP1 shows preferential binding to longer DNA fragments and, in contrast to previous biochemical studies, TopBP1 binding is completely independent of DNA ends. We find that TopBP1 binds to circular and linear DNAs with comparable affinities and that these DNA forms elicit the same level of TopBP1-dependent ATR activation. Taken together, these findings suggest a cooperative activation mechanism for the ATR checkpoint kinase by TopBP1 and damaged DNA

Identification of chromophore binding domains of yeast DNA photolyase

Photolyases contain two chromophores, flavin plus either methenyltetrahydrofolate (MTHF) or 8-OH-5-deazaflavin (HDF). Amino acid sequence comparison reveals that all photolyases sequenced to date have extensive sequence homology in the carboxyl-terminal half; in the amino-terminal region the folate and deazaflavin class enzymes are more homologous to other members of the same class. This modular arrangement of sequence homologies suggests that the amino-terminal half of photolyase is involved in MTHF or HDF binding whereas the carboxyl-terminal half carries the flavin binding site. In this study we attempted to identify such structural domains of yeast photolyase by partial proteolysis and gene fusion techniques. Partial digestion with chymotrypsin yielded an amino-terminal 34-kDa fragment containing tightly bound MTHF and a carboxyl-terminal 20-kDa polypeptide which lacked chromophore or DNA binding activity. However, a fusion protein carrying the carboxyl-terminal 275 amino acids of yeast photolyase bound specifically to FAD but not to MTHF or DNA. We conclude that the amino-terminal half of yeast photolyase constitutes the folate binding domain and that the carboxyl-terminal half carries the flavin binding site

Circadian clock disruption improves the efficacy of chemotherapy through p73-mediated apoptosis

The circadian clock in mammalian organisms is generated by a transcription–translation feedback loop that controls many biochemical pathways at the cellular level and physiology and behavior at the organismal level. Cryptochrome (Cry) is a key protein in the negative arm of the transcription–translation feedback loop. It has been found that Cry mutation in cells with p53-null genotype increased their sensitivity to apoptosis by genotoxic agents. Here we show that this increased sensitivity is due to up-regulation of the p53 gene family member p73 in response to DNA damage. As a consequence, when tumors arising from oncogenic Ras-transformed p53−/− and p53−/−Cry1−/−Cry2−/− cells are treated with the anticancer drug oxaliplatin, p53−/− tumors continue to grow whereas p53−/−Cry1−/−Cry2−/− tumors exhibit extensive apoptosis and stop growing. This finding provides a mechanistic foundation for overcoming the resistance of p53-deficient tumor cells to apoptosis induced by DNA-damaging agents and suggests that disruption of cryptochrome function may increase the sensitivity of tumors with p53 mutation to chemotherapy

Action mechanism of Escherichia coli DNA photolyase. I. Formation of the enzyme-substrate complex.

Escherichia coli DNA photolyase (photoreactivating enzyme) is a flavoprotein. The enzyme binds to DNA containing pyrimidine dimers in a light-independent step and, upon illumination with 300-600 nm radiation, catalyzes the photosensitized cleavage of the cyclobutane ring thus restoring the integrity of the DNA. We have studied the binding reaction using the techniques of nitrocellulose filter binding and flash photolysis. The enzyme binds to dimer-containing DNA with an association rate constant k1 estimated by two different methods to be 1.4 X 10(6) to 4.2 X 10(6) M-1 S-1. The dissociation of the enzyme from dimer-containing DNA displays biphasic kinetics; for the rapidly dissociating class of complexes k2 = 2-3 X 10(-2) S-1, while for the more slowly dissociating class k2 = 1.3 X 10(-3) to 6 X 10(-4) S-1. The equilibrium association constant KA, as determined by the nitrocellulose filter binding assay and the flash photolysis assay, was 4.7 X 10(7) to 6 X 10(7) M-1, in reasonable agreement with the values predicted from k1 and k2. From the dependence of the association constant on ionic strength we conclude that the enzyme contacts no more than two phosphodiester bonds upon binding; this strongly suggests that the pyrimidine dimer is the main structural determinant of specific photolyase-DNA interaction and that nonspecific ionic interactions do not contribute significantly to substrate binding

Regulation of apoptosis by the circadian clock through NF- B signaling

In mice and humans the circadian rhythm of many biochemical reactions, physiology, and behavior is generated by a transcriptional-translation feedback loop (TTFL) made up of the so-called core clock genes/proteins. The circadian system interfaces with most signaling pathways including those involved in cell proliferation and inflammation. Cryptochrome (CRY) is a core clock protein that plays an essential role in the repressive arm of the TTFL. It was recently reported that mutation of CRY in p53-null mice delayed the onset of cancer. It was therefore suggested that CRY mutation may activate p53-independent apoptosis pathways, which eliminate premalignant and malignant cells and thus delay overt tumor formation. Here we show that CRY mutation sensitizes p53 mutant and oncogenically transformed cells to tumor necrosis factor α (TNFα)-initiated apoptosis by interfacing with the NF-κB signaling pathway through the GSK3β kinase and alleviating prosurvival NF-κB signaling. These findings provide a mechanistic foundation for the delayed onset of tumorigenesis in clock-disrupted p53 mutant mice and suggest unique therapeutic strategies for treating cancers associated with p53 mutation

The C-terminal half of UvrC protein is sufficient to reconstitute (A)BC excinuclease.

The UvrC protein is one of three subunits of the Escherichia coli repair enzyme (A)BC excinuclease. This subunit is thought to have at least one of the active sites for nucleophilic attack on the phosphodiester bonds of damaged DNA. To localize the active site, mutant UvrC proteins were constructed by linker-scanning and deletion mutagenesis. In vivo studies revealed that the C-terminal 314 amino acids of the 610-amino acid UvrC protein were sufficient to confer UV resistance to cells lacking the uvrC gene. The portion of the uvrC gene encoding the C-terminal half of the protein was fused to the 3' end of the E. coli malE gene (which encodes maltose binding protein), and the fusion protein MBP-C314C was purified and characterized. The fusion protein, in combination with UvrA and UvrB subunits, reconstituted the excinuclease activity that incised the eighth phosphodiester bond 5' and the fourth phosphodiester bond 3' to a psoralen-thymine adduct. These results suggest that the C-terminal 314 amino acids of UvrC constitute a functional domain capable of interacting with the UvrB-damaged DNA complex and of inducing the two phosphodiester bond incisions characteristic of (A)BC excinuclease