Photoreactivation in phr mutants of Escherichia coli K-12

Abstract

We have investigated the genetics of photoreactivation in Escherichia coli K-12. We found that strains with point mutations or deletions in the phr gene showed a significant residual level of photoreactivation after exposure to large fluences of photoreactivating light. It had been previously proposed that a gene in the gal-attX interval is also involved in photoreactivation and that the residual photoreactivating activity might be due to this so-called phrA gene located at this interval. We found that deletions of the gal-attA region had no effect on either the rate or the final extent of photoreactivation observed in phr+ cells or phr mutants; however strains carrying the A(gal-attf) deletions displayed increased sensitivity to near-UV radiation.Photoreactivation has been defined as the reversal of the effects of far-UV (200-to 300-nm) radiation by subsequent exposure of cells to longer-wavelength light (300 to 600 nm) (7). That the major component of photoreactivation is enzymatic in Escherichia coli was established in 1958 (14), and the first phr mutant was isolated in 1962 (3). This mutation was isolated in E. coli B and subsequently mapped near gal in an E. coli K-12-E. coli B hybrid. (28). Later, it was reported that in E. coli K-12 cells with A(gal-attX), no photoreactivation could be detected, and therefore it was concluded that the gene for photoreactivation, phr, was located in this interval. A Adgal lysogen overproduced a 40-kilodalton (kDa) glycoribonucleoprotein which had photoreactivation activity (21, 22). However, further investigation of the A(gal-attX) mutants showed that these cells were capable of photoreactivating UV damage to the same extent as wild-type cells and that the phr gene was located outside the gal-attA interval, 1 min counterclockwise to gal, between kdp and gltA, and at 16.2 min on the recalibrated E. coli linkage map (1,15,29). The protein encoded by this gene was found to encode a 54-kDa flavoprotein which had in vitro photoreactivation activity comparable to the in vivo rate (17,18). To resolve the controversy, Sutherland and Hausrath (23) investigated the photoreactivation kinetics in wild-type and A(gal-attX) strains and reported that whereas the A(gal-attX) strains could indeed be photoreactivated, the rate of photoreactivation was much slower in strains with this deletion; they suggested that there were two genes controlling photoreactivation in E. coli, one at the gal-attX interval and one at the kdp-gltA interval, and proposed the designations phrA and phrB, respectively, for these loci. More recently it has been suggested (24) that the phrA gene encodes a 40-kDa protein that has an RNA cofactor (21), while the phrB gene encodes the 54-kDa photolyase that has a flavin adenine dinucleotide cofactor (17). However, the presence of two photoreactivating enzymes is inconsistent with other genetic data indicating that Harm's phr (phrB) mutant cannot be photoreactivated (2, 3), as well as with biochemical data showing the lack of dimer splitting by cell extracts from this st...

A cryptochrome/photolyase class of enzymes with single-stranded DNA-specific photolyase activity

“…The results in Fig. 2 show that whereas photolyase demonstrates some preference for single-stranded over double-stranded substrates, in agreement with previous reports (17,18), VcCry1 unequivocally repairs the dimer but only in ssDNA. Furthermore, the other Cry-DASH proteins from plant (AtCry3) and animal (XlCry-DASH) sources also demonstrated photoreactivation specifically toward ssDNA.…”

“…It should be noted, however, that this classification does not necessarily exclude an additional nonrepair function for ssDNA photolyases, as indeed even some conventional photolyases have both repair and transcriptional regulatory functions (21). With regard to single-strand specificity of this class of enzymes, it must be noted that both biochemical (17) and crystallographic (22,23) data indicate that conventional photolyases make most of their contacts with the damaged strand and the contacts with the undamaged strand are mostly van der Waals contacts of secondary importance. It appears that this preference for the damaged strand is amplified in this new family of photolyases by the presence of amino acid residues in the binding groove that clash with the phosphodiester backbone or the bases of the complementary strand.…”

Dna Repair Enzymes

“…Sancar, unpublished], similar to the rate observed in vivo (13 ). Reports of an RNA-containing photolyase encoded by a gene in the gal-atOl interval (20) have been shown to be in error (21). The phr gene is not essential for viability; however phr-mutants, in addition to being unable to photoreactivate, are also partly deficient in excision repair of pyrimidine dimers (22,23) because the enzyme stimulates ABC excinuclease (24).…”

“…Begley, G. Payne, A. Sancar, unpublished). The precise contacts made on DNA by photolyase have been mapped using as substrate a 43-bp DNA duplex containing a single pyrimidine dimer at a unique location (37). Enzymatic and chemical probes reveal that: photolyase interacts with a 6--7-bp region around the dimer; intimate contact between enzyme and DNA occurs only on the damaged strand.…”

Structure and Function of Photolyase and in Vivo Enzymology: 50th Anniversary

“…These features of the DNA backbone are recognized by photolyase, which makes a moderately stable complex through ionic interactions between the positively charged groove on the surface of the enzyme and the first phosphate 5Ј and the three phosphates 3Ј to the TϽϾT on the damaged strand and some weaker interactions with the backbone of the complementary strand across from the dimer (24). These interactions further weaken the duplex in the immediate vicinity of the TϽϾT, leading to "flipping out" of the TϽϾT into the active-site cavity in the middle of the DNA binding groove (20) to make a high stability complex.…”