Michael H. Patrick

Near-U.V. photolysis of pyrimidine heteroadducts in E. coli DNA.

Abstract— The cytosine‐thymine precursor of the U–T adduct is not subject to enzymatic photoreactivation, but can be eliminated directly from u.v.‐irradiated E. coli DNA by exposure to wavelengths around 313 nm.

Independence of photoproduct formation on DNA conformation.

Abstract— In an ethanolic solution native T7 DNA can undergo conformational transitions from the B conformation (0% ethanol) to the C‐like (60% w/w ethanol) and the A (80% w/w ethanol) conformations. We have investigated the formation of three classes of thymine‐derived photoproducts in T7 DNA irradiated (280 nm) in the B, C‐like, and A conformations, which were monitored by circular dichroism measurements. We find that the predominant class of thymine‐derived photoproducts in any conformational state is cyclobutyl dipyrimidines. While the ‘spore product,’ 5‐thyminyl‐5,6‐dihydrothymine, which belongs to another class of photoproductsf does form in native DNA in the A conformation, its yield in denatured DNA at 80% ethanol is the same as that in native DNA. The yield of pyrimidine adduct, a third photoproduct class, is a maximum at 50–60% ethanol. This effect of ethanol is probably not due to the ethanol‐induced C‐like conformation, however, since pyrimidine adduct formation is not enhanced when T7 DNA is irradiated in the C conformation in 6 M CsCl or in intact phage. We conclude from these and other data in the literature that the degree of hydration rather than the conformational state is the critical factor in determining which of the photoproducts will form in native DNA.

ACTION OF PHOTOREACTIVATING LIGHT ON PYRIMIDINE HETEROADDUCT IN BACTERIA

Abstract— In stationary‐phase Escherichia coli B/r, photoreactivation (PR) at 313 nm of ultraviolet (u.v.) killing is inefficient compared with PR at 405 nm, and can be explained solely by photoenzymatic reversal of pyrimidine dimers.

PHOTOREACTIVATION OF KILLING IN STREPTOMYCES-III. ACTION SPECTRA FOR PHOTOLYSIS OF PYRIMIDINE DIMERS AND ADDUCTS IN S. GRISEUS AND S. GRISEUS PHR-l*

Abstract— Photolysis of tritium‐labelled thymine‐derived photoproducts by 254‐nm ultraviolet radiation (u.v.) in conidia of Streptomyces griseus was measured by chromatography of cell hydrolysates. The relative photolysis cross‐sections of uracilthymine dimer (UT○) at various wavelengths are the same as those of thymine‐thymine dimer (TT○), and their ratios at 313, 365, 405 and 436 nm are 2:1:2:3. Except at 436 nm, these relative values agree very well with cross‐sections previously reported for photoreactivation of u.v. killing in this organism, leading to the conclusion that photoreactivation in the wild type is due to repair of cyclobutane‐type pyrimidine dimers. In a mutant showing restricted photoreactivation (S. griseus PHR‐1), post‐u.v. treatments at the above wavelengths did not affect UT○ and TT○ in the conidia, supporting the earlier suggestion that this organism does not contain active PR enzyme. Another u.v. photoproduct, the precursor of a pyrimidine adduct (PO‐T) that appears in cell hydrolysates, was removed from both wild‐type and mutant cells very efficiently at 313 nm. This is presumably a direct photochemical reaction. In addition, in wild‐type cells, the precursor of PO‐T appeared to be inefficiently removed photoenzymatically at all wavelengths. Removal of the precursor of PO‐T appears to be biologically significant, however, only in the mutant.

STUDIES ON THYMINE-DERIVED UV PHOTO-PRODUCTS IN DNA—II. A COMPARATIVE ANALYSIS OF DAMAGE CAUSED BY 254 NM IRRADIATION AND TRIPLET-STATE PHOTOSENSITIZATION

Abstract— From the rates of cyclobutyl dipyrimidine (Pyr < > Pyr) formation and the ratio of inactivation of transforming or phage DNA caused by direct (254 nm) or sensitized (1.0 M acetone, 313 nm; 0.02 M acetophenone, 334 nm) irradiation, we conclude that Thy < > Pyr and Cyt < > Pyr are equally lethal, and that they are repaired with equal efficiency by the host cell. Not all the damage formed by photosensitized irradlation can be photoenzymatically repaired, especially when acetone is the sensitizer. We found no compelling evidence for photosensitized interstrand cross‐links or sensitizer‐DNA addition products for the fluence range used in these studies (< 106 Jm‐2); moreover, strand breakage can account for only a part of the non‐photorepairable damage. We suggest that a fraction of the damage may be due to Pyr < > Pyr isomers other than the cis, syn type usually formed in native DNA by far‐UV light.

REPAIR OF UV DAMAGE IN ESCHERICHIA COLI UNDER NON-GROWTH CONDITIONS

Abstract. –A large difference in survival occurs between buffered suspensions of E. coli irradiated with UV radiation at a low fluence rate and those irradiated at a high fluence rate. For sufficiently large fluences, the extent of this fluence rate dependent recovery (FRR) is about two orders of magnitude greater than that which can be brought about by liquid holding recovery (LHR) following high fluence rate irradiation in most of the E. coli strains studied. LHR and FRR occur in excision resynthesis repair proficient (ERR+) but not ERR‐ strains of E. coli, although its observation can be masked in strains with complete repair potential upon subsequent growth on nutrient plates. Accumulation of DNA strand interruptions and excision of cyclobutyl dipyrimidine occur during LHR and FRR but are more extensive for the latter. Our data suggest mat events beyond incision and excision occur during LHR and FRR, but differences in the extent of ERR during LHR and FRR cannot account for the difference in cell survival between these two phenomena.

SUBSTRATE SPECIFICITY OF A BACTERIAL UV ENDONUCLEASE AND THE OVERLAP WITH IN VITRO PHOTOENZYMATIC REPAIR

Abstract— The action of an endonuclease from Micrococcus luteus, that operates on ultraviolet (UV) radiation damage, overlaps greatly with that of the yeast photoreactivating enzyme: homo and hetero cyclobutyl pyrimidine dimers in DNA are substrate for both enzymes, but pyrimidine adducts or the ‘spore photoproduct’ in DNA are not.

DNA turnover in buffer-held Escherichia coli and its effect on repair of UV damage.

Abstract— Continuous DNA degradation and resynthesis, without a net change in cellular DNA content, were observed in buffer‐held, non‐irradiated E. coli B/r. This constant DNA turnover probably involves most of the genome and reflects random sites of DNA repair due to the polA‐dependent excision‐resynthesis repair pathway. Under these non‐growth conditions, it appears that at any given time there is a minimum of one repair site per 6.5 × 106 daltons DNA, each of which is at least 160 nucleotides long.

The role of DNA polymerase I in liquid holding recovery of UV-irradiated Escherichia coli.

Abstract. –Excision of cyclobutyl dipyrimidines from, and accumulation of strand interruptions in, DNA of different strains of E. coli K12 were determined during liquid holding recovery after UV irradiation. The extent of Pyr <> Pyr excision was the same (20–25%) for both a polA mutant (E. coli P3478) and its parental wild type strain (E. coli W3110); however, single strand interruptions accumulate during liquid holding of polA cells, but not in the parental strain. In contrast, excision was greatly reduced in a mutant (KMBL 1789) which is defective in the 5′→3′ exonucleolytic function of DNA polymerase I. These data suggest that excision and resynthesis during liquid holding are carried out primarily, if not entirely, by DNA polymerase I. We further conclude that excision alone is both a necessary and sufficient condition to elicit liquid holding recovery, and that this excision requires a functional polymerase I 5′→ 3′ exonuclease.

Examination of newly synthesized DNA in Escherichia coli

SummaryThe short (0-20S) “Okazaki” fragments seen upon pulse-labeling E. coli (thy+, deo+) with 3H-thymidine are actually composed of three types of DNA fragments: (1) true replication intermediates, (2) post-replication repair fragments (such as those which arise subsequent to the removal of misincorporated uracil), and (3) chromosomal fragments. Our experiments show that the number of pulse-labeled fragments decreases slightly with the introduction of the ung-1 mutation into E. coli K-12 (dut+, thyl+, polA+), and that there are fewer fragments found in E. coli B/r than in E. coli K-12 ung-1. This suggests that while some fraction of pulse-labeled fragments may be due to repair, this fraction can vary among different strains; moreover, the majority of fragments appear to be replication intermediates. Selfhybridization (reannealing) of pulse-labeled fragments from E. coli B/r show that these fragments are asymmetric with respect to the strand origin and with respect to their size: the smaller (3-8S) fragments come from only one of the parental strands, whereas the larger (13-20S) fragments are synthesized equally from both parental strands. We interpret our results to mean that replication can be discontinuous on both strands but asymmetric with respect to both the size of the fragments and the size of the discontinuous region on the two strands.The short (0-20S) “Okazaki” fragments seen upon pulse-labeling E. coli (thy+, deo+) with 3H-thymidine are actually composed of three types of DNA fragments: (1) true replication intermediates, (2) post-replication repair fragments (such as those which arise subsequent to the removal of misincorporated uracil), and (3) chromosomal fragments. Our experiments show that the number of pulse-labeled fragments decreases slightly with the introduction of the ung-1 mutation into E. coli K-12 (dut+, thyl+, polA+), and that there are fewer fragments found in E. coli B/r than in E. coli K-12 ung-1. This suggests that while some fraction of pulse-labeled fragments may be due to repair, this fraction can vary among different strains; moreover, the majority of fragments appear to be replication intermediates. Selfhybridization (reannealing) of pulse-labeled fragments from E. coli B/r show that these fragments are asymmetric with respect to the strand origin and with respect to their size: the smaller (3-8S) fragments come from only one of the parental strands, whereas the larger (13-20S) fragments are synthesized equally from both parental strands. We interpret our results to mean that replication can be discontinuous on both strands but asymmetric with respect to both the size of the fragments and the size of the discontinuous region on the two strands.