The incision and strand rejoining step in the excision repair of 5,6-dihydroxy-dihydrothymine by crude E. coli extracts

Strand resealing in the $\text{in}$$\text{vitro}$ excision repair of 5,6-dihydroxy-dihydrothymine in osmium tetroxide oxidized polyd(A-T) by crude $\text{E.}$$\text{coli}$ extracts is accomplished by polynucleotide ligase. Osmium tetroxide oxidized polyd(A-T)_serves as a chemically well defined model substrate containing damage of the kind introduced into DNA by ionizing radiation. In the first incision step of excision repair approximately one endonucleolytic nick is introduced into the polymer by extracts of $\text{E.}$$\text{coli}$ endoI^? and $\text{E.}$$\text{coli}$ endoI^?$\text{uvr}$A6^? per ring damaged thymine residue removed.     

Repair of mitomycin C damage to DNA in mammalian cells and its impairment in Fanconi's anemia cells.

Repair of DNA cross-links by mitomycin C (MMC) was studied in mammalian cells. Skin cells from a patient with Fanconi's anemia (FA9 cells) were about 6 times as sensitive to MMC killing as HeLa S3 cells with normal excision repair ability, while excision-reduced mouse L and human xeroderma pigmentosum (XP2OS) cells were more resistant to it than HeLa S3 cells. Alkaline sucrose sedimentation of DNA revealed that perhaps half-excision of cross-links and its repair occurred efficiently until 4 h of post-MMC time in L-cells and, though more slowly, in HeLa S3 cells. Thus, the excision repair pathway is the first step of the cross-link repair in mammalian cells, but it seems different from the $\text{uvrA}$-dependent pathway in $\text{E. coli}$, since XP2OS cells survived MMC almost normally. Contrarily, FA9 DNA sedimented much faster at 4 h of post-MMC time, suggesting a possible impairment in FA cell's ability to unhook cross-links.     

Excision repair of ultraviolet radiation damage in toluene treated Escherichia coli

ATP independent excision repair of UV damage has been studied in $\text{E. coli}$ made permeable to nucleotides by treatment with toluene. In using this system, separation of the first step from the subsequent steps in the repair process is achieved. It was found that completion of repair is observed only in strains that have normal levels of DNA polymerase I.     

Nature of the repair process associated with the recovery of Escherichia coli after exposure to Cd2+.

When different strains of $\text{Escherichia coli}$ are exposed to Cd²⁺, the cells accommodate after a long lag and proliferate. The time required for this response depends on the nature of the strain and the supplements in the growth medium. Immediately after exposure to Cd²⁺, considerable single strand breaks in the DNA are observed but the DNA is repaired prior to the initiation of cell proliferation. The finding that accommodation occurs in DNA polymerase I-deficient mutant cells suggests that DNA polymerase I may not be required for repair of damaged DNA in Cd²⁺-exposed cells. The recovery of Cd²⁺-exposed cells in a temperature-sensitive DNA ligase mutant cells at the permissive temperature (30° C) and failure to recover at the non-permissive temperature (42° C) indicates, however, that DNA ligase is involved in the repair of the single strand breaks associated with Cd²⁺-induced damage.     

The modifying effect of sodium ascorbate on DNA damage and repair after N-methyl-N′-nitro-N-nitrosoguanidine treatment in vivo

A biological reducing agent, sodium ascorbate, was used to modify both the damage induced by N-methyl-N′-nitro-N-nitrosoguanidine to mouse gastric mucosal cell DNA and the repair of that damage $\text{in vivo}$. Freshly-mixed carcinogen and sodium ascorbate enhanced DNA fragmentation as measured by shifts in alkaline sucrose gradient sedimentation profiles whereas incubation of the two compounds for a short period resulted in reduced DNA fragmentation. Furthermore, periodic administration of sodium ascorbate following stomach cell DNA damage with carcinogen inhibited DNA repair.     

DNA postreplication repair gap filling in temperature-sensitive dnaG, dnaC, dnaA mutants of Escherichiacoli

Gaps in daughter-strand deoxyribonucleic acid (DNA) synthesized after exposure of wild-type $\text{Escherichia}\text{coli}$ to ultraviolet light are filled during reincubation. In this study the $\text{dnaG}\text{,}\text{dnaC}$, and $\text{dnaA}$ gene products have been examined for their role in postreplication repair. These gene products are unique in their specific control of certain types of DNA synthesis: initiation of rounds of replication and chain propagation. Initiation of rounds of replication is not essential to gap filling; however, chain propagation by short DNA piece initiation appears to be essential for gap filling.     

Effect of aphidicolin on viral and human DNA polymerases.

DNA polymerases induced by Herpes simplex and Vaccinia viruses are inhibited by aphidicolin and this inhibition is probably the basis of its antiviral activity $\text{in vivo}$. Its possible clinical use is however hampered by the concomitant effect on human replicative DNA polymerase α. The inhibition of human α-polymerase is reversible both $\text{in}$$\text{vitro}$ and $\text{in vivo}$ and the changes in the rate of incorporation of thymidine into DNA, following treatment with aphidicolin for a generation time, indicate the likely synchronization of the cells due to this agent. DNA polymerase β, which has recently been shown to carry out repair synthesis of damaged nuclear DNA, is not inhibited by aphidicolin either $\text{in vitro}$ on $\text{in vivo}$ suggesting that the drug could allow a rapid and simple evaluation of DNA repair synthesis due to DNA polymerase β.     

Alternative Excision Repair Pathways

Alternative excision repair (AER) is a category of excision repair initiated by a single nick, made by an endonuclease, near the site of DNA damage, and followed by excision of the damaged DNA, repair synthesis, and ligation. The ultraviolet (UV) damage endonuclease in fungi and bacteria introduces a nick immediately 5′ to various types of UV damage and initiates its excision repair that is independent of nucleotide excision repair (NER). Endo IV-type apurinic/apyrimidinic (AP) endonucleases from Escherichia coli and yeast and human Exo III-type AP endonuclease APEX1 introduce a nick directly and immediately 5′ to various types of oxidative base damage besides the AP site, initiating excision repair. Another endonuclease, endonuclease V from bacteria to humans, binds deaminated bases and cleaves the phosphodiester bond located 1 nucleotide 3′ of the base, leading to excision repair. A single-strand break in DNA is one of the most frequent types of DNA damage within cells and is repaired efficiently. AER makes use of such repair capability of single-strand breaks, removes DNA damage, and has an important role in complementing BER and NER.NER and base excision repair (BER) are the major excision repair pathways present in almost all organisms. In NER, dual incisions are introduced, the damaged DNA between the incised sites is then removed, and DNA synthesis fills the single-stranded gap, followed by ligation. In BER, an AP site, formed by depurination or created by a base damage-specific DNA glycosylase, is recognized by an AP endonuclease that introduces a nick immediately 5′ to the AP site, followed by repair synthesis, removal of the AP site, and final ligation. Besides these two fundamental excision repair systems, investigators have found another category of excision repair—AER—an example of which is the excision repair of UV damage, initiated by an endonuclease called UV damage endonuclease (UVDE). UVDE introduces a single nick immediately 5′ to various types of UV lesions as well as other types of base damage, and this nick leads to the removal of the lesions by an AER process designated as UVDE-mediated excision repair (UVER or UVDR). Genetic analysis in Schizosaccharomyces pombe indicates that UVER provides cells with an extremely rapid removal of UV lesions, which is important for cells exposed to UV in their growing phase.Endo IV–type AP endonucleases from Escherichia coli and budding yeast and the Exo III–type human AP endonuclease APEX1 are able to introduce a nick at various types of oxidative base damage and initiate a form of excision repair that has been designated as nucleotide incision repair (NIR). Endonuclease V (ENDOV) from bacteria to humans recognizes deaminated bases, introduces a nick 1 nucleotide 3′ of the base, and leads to excision repair initiated by the nick. These endonucleases introduce a single nick near the DNA-damage site, leaving 3′-OH termini, and initiate repair of both the DNA damage and the nick. The mechanisms of AER may be similar to those of single-strand break (SSB) repair or BER except for the initial nicking process. However, how DNA damage is recognized determines the repair process within the cell. This article discusses the mechanisms and functional roles of AER. We begin with AER of UV damage, because genetic analysis has shown functional differences between this AER and NER in S. pombe.     

Bacillus subtilis SbcC protein plays an important role in DNA inter-strand cross-link repair

Background  

Several distinct pathways for the repair of damaged DNA exist in all cells. DNA modifications are repaired by base excision or nucleotide excision repair, while DNA double strand breaks (DSBs) can be repaired through direct joining of broken ends (non homologous end joining, NHEJ) or through recombination with the non broken sister chromosome (homologous recombination, HR). Rad50 protein plays an important role in repair of DNA damage in eukaryotic cells, and forms a complex with the Mre11 nuclease. The prokaryotic ortholog of Rad50, SbcC, also forms a complex with a nuclease, SbcD, in Escherichia coli, and has been implicated in the removal of hairpin structures that can arise during DNA replication. Ku protein is a component of the NHEJ pathway in pro- and eukaryotic cells.     

Demethylation of O6-methylguanine in a synthetic DNA polymer by an inducible activity in Escherichiacoli

$\text{O}$⁶-Methyl[8-³H]deoxyguanosine in a synthetic DNA polymer, poly(dC, dG, m⁶dG), is demethylated by cell-free extracts of $\text{Escherichia}\text{coli}\text{B}\text{r}$ adapted by exposure to $\text{N}$-methyl-$\text{N}$′-nitro-$\text{N}$-nitrosoguanidine, as shown by the appearance of ³H-labeled deoxyguanosine in hydrolysates of the recovered DNA. The demethylating activity could not be detected in extracts of nonadapted $\text{E}\text{.}\text{coli}$. These results provide direct evidence that a previously described inducible repair activity in $\text{E}\text{.}\text{coli}$ acts by demethylating $\text{O}$⁶-methylguanine at the DNA level.     

Genetic transformation in E.coli: The inhibitory role of the recBC DNase

Genetic transformation of $\text{E.}\text{coli}$ for various chromosomal markers was accomplished by (i) using recipient cells that lack the $\text{recBC}$ DNase but were recombination proficient due to $\text{sbcA}$ or $\text{sbcB}$ mutations and (ii) treating the recipient cells with CaCl₂ at a concentration that facilitates transfection by λ DNA. Cotransformation of three markers ($\text{thr}{}^{\mathrm{+}}\text{ara}{}^{\mathrm{+.}}\text{leu}{}^{\mathrm{+}}$) was found to depend on the molecular weight of the transforming DNA.     

The stability of deoxycytidine photohydrates in the mononucleotide, oligodeoxynucleotides and DNA

The stability of deoxycytidine photohydrates was determined for deoxycytidylic acid and deoxycytidine residues in oligodeoxynucleotides by optical measurements and in native and denatured DNA by a chemical assay. The half lives at 20° in $\text{10}{}^{\mathrm{?2}}\text{M}$ tris buffer, pH 7.7, were 102 min. for the mononucleotide, 128 min. for dpApGpG, 152 min. for MeOdpTpCpA, 51 min. for denatured $\text{E}\text{.}\text{coli}\text{DNA and 58 min. for native}\text{E}\text{.}\text{coli}$ DNA (at pH 8.1). It is concluded that the stability of deoxycytidine photohydrates is sufficient that they cannot at present be dismissed as lesions of possible biological importance in ultraviolet irradiated cells.     

Genetic and enzymatic characterization of a conditional lethal mutant of Escherichia coli K12 with a temperature-sensitive DNA ligase

$\text{TAU}$ts7 an Escherichia coli 15 strain with a thermolabile DNA ligase, has previously been shown to be a temperature-sensitive conditional lethal mutant that is sensitive to methyl methane sulfonate and to ultraviolet irradiation; it also accumulates 10 S DNA fragments to an abnormal extent. When the ligase mutation is transferred to a wild-type E. coli K12 strain, the strain becomes temperature sensitive for growth and displays the same characteristics as $\text{TAU}$ts7. These findings show that a functional DNA ligase is essential for normal DNA replication and repair in E. coli.     

The specific uptake of cloned Haemophilus DNA.

During the process of transformation $\text{Haemophilus}\text{influenzae}$ cells bind its own DNA but little or no foreign DNA. This specificity for recognition of DNA was studied by cloning Haemophilus DNA in $\text{E. coli}$. Haemophilus DNA fragments were cloned using plasmid pBR322 as a vector. The fragment cH7 cloned in pBR322 was found to be homologous to Haemophilus DNA and shown to bind irreversibly to competent Haemophilus cells. The fact that cH7 isolated from $\text{E. coli}$ lacks Haemophilus modification leads to the conclusion that modification does not play a role in the uptake mechanism. Uptake specificity is a function of recognition sequences that reside in DNA itself.     

Ultraviolet light-induced recombination

Stimulation of transduction in $\text{Escherichia coli}$ by ultraviolet irradiation of the transducing phage P1 requires the $\text{uvrA-uvrB}$ nuclease but not the $\text{uvrC}$ product or DNA polymerase I. It is hypothesized that the first step in “normal” recombination can be bypassed by any procedure generating single-stranded ends of DNA (as, for example, by $\text{uvra-uvrB}$ nuclease activity).     

The effect of 5-azacytidine on E. coli DNA methylase.

5-Azacytidine, when added to growing $\text{E.}$$\text{coli}$ K12, causes a decrease in DNA methylation assayed $\text{in}$$\text{vitro}$. This decrease is greater when $\text{E.}$$\text{coli}$ DNA is used as substrate than when calf thymus DNA is used. The decrease in activity is not due to the inhibition of protein synthesis caused by this drug, since neither chloramphenicol nor rifampin causes a decrease in enzyme activity. The effect is specific for the DNA(cytosine-5)methylase; the methylation of adenine is not affected. The concentration of drug that inhibits the DNA methylase by 50% is the same concentration that inhibits cell growth by 50%.     

Common precursors of human blood group MN specificities

Human blood group MM and NN specific structures have the same precursors. Complete sialic acid removal produced the Thomsen-Friedenreich T antigen which was transformed into Tn antigen by $\text{E. coli}\text{β-}\text{D}$-galactosidase on red cells as well as on isolated T antigen. MN antigens and their precursors are most clearly defined by isologous human antisera.