Genetics and Developmental Biology

The Thomas Hunt Morgan Centennial Symposium

Howard J. Teas

• Publisher: The University Press of Kentucky
• Year: 2021
• Print ISBN:
  9780813154961
• eBook ISBN:
  9780813183541
• Topic: Biological Sciences
• Subtopic: Biology
• Index: Biological Sciences

GENETIC REPAIR MECHANISMS

Paul Howard-Flanders

Because of the unique and dominant role played by the genome, the cell is probably more affected by any disruption of covalent bonds in its genome than in any other cell constituent. Any irreparable damage to the DNA thread may result in a loss of function and lead to failure in cell reproduction and to cell death. It is hardly surprising, therefore, that cells should have evolved special devices for the preservation of the genome in adverse circumstances, such as when subject to damage by chemical mutagens, or high-energy radiations.

Several mechanisms for reconstructing damaged or fragmented genomes have been discovered in Escherichia coli, Bacillus subtilis, and Micrococcus lysodeikticus.

Notable advances into this field followed investigations into the effects of ultraviolet (UV) light and into the manner in which cells respond to the formation of UV photoproducts in their DNA. Studies with UV light have been profitable, because this agent is known to form specific photoproducts in the DNA of the cell, which are stable and can be measured chemically. In this paper, after briefly summarizing the properties of the UV photoproducts and two well-known mechanisms for survival after UV irradiation, a third and possibly more versatile mechanism will be described.

UV Photoproducts in DNA

Exposure to UV light induces the formation of various photoproducts in DNA (Beukers, Ijlstra, and Berends, 1960; Wacker, 1963; Smith, 1964; Setlow, J. K., 1966). Dimers are former between adjacent pyrimidine bases in high yield and comprise 50 percent thymine-thymine dimers, 40 percent thymine-cytosine dimers, while cytosine-cytosine dimers represent only 10 percent of the yield (Setlow, R. B., Carrier, and Bollum, 1965; Setlow, R. B., 1966). These dimers are formed by linking the unsaturated double bonds to form a cyclobutane ring, and they must cause considerable local distortion of the phosphodiester backbone of the twin helix. The principal thymine-thymine dimer in UV-irradiated DNA is linked in the 5-5 and 6-6 positions in a cis-syn boat configuration, as would be expected if it is formed from adjacent bases in the same single strand of a twin helix. This isomer can be distinguished from others by chemical means (Blackburn and Davies, 1966). Pyrimidine dimers are probably the major products formed by UV light in DNA, but other products such as hydrates are formed in lesser amounts. As the yield of dimers is known, a defined number of these stable defects can be produced in DNA at will by exposing cells, or DNA, to the appropriate dose of UV light. The yield of pyrimidine dimers is about 1.3 x 10^–6 dimers per base pair per erg/mm² at 2537 A (Setlow, R. B., 1966; Wulff, 1963; Setlow, R. B., Swenson, and Carrier, 1963; Boyce and Howard-Flanders, 1964a). A UV dose of 1 erg/mm² will produce about six dimers in a genome of 10⁷ nucleotides (which is about the number of nucleotides in the genome of E. coli deduced by autoradiography (Cairns, 1963).

The biological effects of UV light are due, at least in part, to the formation of pyrimidine dimers and other photoproducts in DNA (Setlow and Setlow, 1962). Just how much of any biological effect is due to a particular type of radiation product is not readily determined because of the difficulty of producing only specific products in particular components of the cell. Moreover, many of the biological effects may be due to products formed in DNA, which are subject to the action of repair enzymes.

Fortunately for the development of the subject, pyrimidine dimers can be measured chemically and are stable. No less than three mechanisms are known for obviating the effects of such dimers in E. coli. These are: (a) photoreactivation, (b) excision, and (c) exchanges between sister duplexes following DNA replication. All three mechanisms can be effective in promoting colony formation in cells exposed to UV light.

Photoreactivation

When exposed to UV irradiation, many microorganisms exhibit a loss of viability and other biological functions. Partial recovery of biological activity may occur if the cells are subsequently exposed to light of about 3,500

wavelength. Photoreactivation, as this is called, is believed to reflect the action of a photoreactivating enzyme which has been extracted and purified (Rupert, 1962; Mohammed, 1966). If DNA containing pyrimidine dimers is treated with photoreactivating enzymes and exposed to 3,500

light, the dimers are removed and probably monomerized in situ (Setlow, R. B., Carrier, and Bollum, 1965; Setlow, J. K., Boling, and Bollum, 1965; Cook, 1967). Also of interest is the isolation of a mutant of E. coli defective in photoreactivation (Harm and Hillebrandt, 1962) and the finding that this strain lacks active photoreactivating enzyme (Rupert, 1965). It may be significant to understanding this phenomenon that photoreactivation can be mimicked by dye-sensitized photodissociation of thymine dimers in solution if a suitable dye is present (Lamola; 1966). This dissociation may reflect the transfer of energy from a triplet excited state in the dye to the dimer.

DNA Repair by Excision of Defects

After exposure to UV light, the repair of DNA is initiated by the excision of pyrimidine dimers. Both thymine-thymine and thymine-cytosine dimers are released or excised from the DNA of certain microorganisms during incubation following irradiation. Pyrimidine dimers are released in wild type but not in certain UV-sensitive mutants, and they are recovered within acid-soluble oligonucleotides. Evidently, dimers are released from the DNA by excising short single-strand fragments (Boyce and Howard-Flanders, 1964a; Setlow, R. B. and Carrier, 1964). This release of dimers into an acid-soluble form has been reported in E. coli, Micrococcus radiodurans, Bacillus megaterium, T4-phage-infected E. coli, and B. subtilis (Setlow, R. B., 1966; Strauss, Searashi, and Robbins, 1966; Shuster, 1967). Evidence for an in vitro enzyme activity for the release of pyrimidine dimers from DNA has been obtained in extracts of M. lysodeikticus and there has been some success in the purification of a dimer-specific endonuclease (Carrier and Setlow, 1966; Miller, et al., 1967). An enzyme activity which may be similar but was detected in extracts of the same organism by a biological assay has also been reported (Rorsch, Kamp, and Adema, 1964; Elder and Beers, 1965). Although these systems have been used to demonstrate the excision of dimers in vitro, further purification and investigation of the characteristics of the excision enzyme are still necessary.

The evidence that excision may constitute the first step in a process for the repair of DNA-containing dimers depends upon the properties of UV-sensitive mutants lacking the ability to release dimers, that have been isolated in several organisms. The greater sensitivity of these strains may reflect their inability to initiate DNA repair by the excision of the dimers. These excision-defective mutants are abnormally sensitive not only to UV light but also to bifunctional alkylating agents (Haynes, 1964), mitomycin C (Boyce and Howard-Flanders, 1964b), and nitrous acid (Finesilver, 1968). The range of mutagens to which these excision-defective strains are abnormally sensitive is much wider than the range for which evidence of the photoreactivation of treated cells can be obtained. An interpretation of this difference in specificities is that whereas the photoreactivation may act by an enzyme-sensitized photodissociation and act only on a restricted class of structures, the excision enzyme acts by cutting covalent bonds in the phosphodiester backbone on either side of the damaged bases. To judge by its more catholic range of action, the excision enzyme may recognize a distortion of the DNA twin helix near the site of the damage rather than the specific product responsible. It may be significant that all the mutagens whose effects are more pronounced in excision-defective than in normal cells are cross-linking agents (Haynes, 1964; Boyce and Howard-Flanders, 1964b; Finesilver, 1968). Much interesting work remains to be done in exploring the sterio-chemical specificities of excision enzymes.

DNA Degradation and Repair Replication

When E. coli is UV irradiated and incubated, there is a degradation of the DNA in wild-type strains, but not in excision-defective mutants. This breakdown may be initiated by excision and reflect the action of an enzyme on the free single strands ending at these cuts. The total number of nucleotides released may be several hundred times larger than the number of dimers initially present in the DNA (Boyce and Howard-Flanders, 1964b; Howard-Flanders and Boyce, 1966). This suggests that the gaps left by excision may be substantially widened.

If the repair is to be completed, the gap must be filled with nucleotides complementary to those of the intact opposite strand, a process called repair replication. Evidence for this has been obtained, as radioactive, or density-labeled, nucleotides are incorporated into small patches in the DNA of UV-irradiated cells, rather than being restricted to the region of new DNA synthesis as in normal replication (Pettijohn and Hanawalt, 1964; Hanawalt, 1967; Billen et al., 1967). Repair will be completed when the phosphodiester backbone is joined following the insertion of the last nucleotide into the gap.

The properties of certain enzymes of E. coli may be relevant to understanding the mechanism of DNA repair. Exonuclease III is effective on two-strand DNA and releases nucleotides from the 3′ terminus (Richardson and Kornberg, 1964). E. coli DNA polymerase acts by the addition of 5′ nucleotides to the 3′ terminus (Richardson, et al., 1964). These enzymes have the specificities required for single-strand breakdown and repair synthesis and have been used to demonstrate the removal of nucleotides and the repair of λ-phage DNA (Richardson, Inman, Kornberg, 1964). However, there is no evidence to show whether these are the enzyme responsible for DNA degradation and repair synthesis in E. coli. If they are, it seems likely that the single-strand gaps formed by dimer excision are widened and then filled in by action on the 3′ side of the gap. However, a direct test is still needed to show whether DNA breakdown and repair synthesis following excision proceeds in this fashion.

The formation of single-strand gaps and their subsequent repair in UV-radiated E. coli has been observed in experiments on the sedimentation of bacterial DNA in alkaline sucrose. Even when the DNA from a cell contains several thousand dimers, fewer than twenty, single-strand gaps, appear to be open at any one time (Setl...

Table of contents

• Cover
• Half title
• Title
• Copyright
• Preface
• Contents
• Introduction
• Nonrandom Segregation of Sister Chromatids
• Meiosis and DNA Replication in Chlamydomonas
• Genetic Repair Mechanisms
• Chromosome Transfer in Bacterial Mating
• Suppression of Phage Mutants by Ochre Suppressors
• Gene Action in the Control of Bacteriophage T4 Morphogenesis
• Mitochondrial Transfer RNA’s and Aminoacyl-RNA Synthetases
• ‘Masked’ Messenger RNA and the Determination Process in Embryonic Development
• The Control of Genetic Activity
• Replication of Chromosomal DNA and Mechanisms of Recombination
• Contributors