Deoxyribonucleic acid repair in vitro by extracts of Escherichia coli.

Deoxyribonucleic acid (DNA) from bacteriophage T7 has been used to monitor the capacity of gently lysed extracts of Escherichia coli to perform repair resynthesis after ultraviolet (UV) irradiation. Purified DNA damaged by up to 100 J of UV radiation per m2 was treated with an endonuclease from Micrococcus luteus that introduces single-strand breaks in irradiated DNA. This DNA was then used as a substrate to study repair resynthesis by extracts of E. coli. It was found that incubation with the extract and exogenous nucleoside triphosphates under suitable assay conditions resulted in removal of all pyrimidine dimers and restoration of the substrate DNA to its original molecular weight. Repair resynthesis, detected as nonconservative, UV-stimulated DNA synthesis, was directly proportional tothe number of pyrimidine dimers introduced by radiation. The repair mode described here appears to require DNA polymerase I since it does no occur at the restrictive temperature in polA12 mutants, which contain a thermolabile polymerase. The addition of purified DNA polymerase I to extracts made from a polA mutant restores the ability to complete repair at the restrictive temperature.     

The 5'' to 3'' exonuclease activity of DNA polymerase I is essential for Streptococcus pneumoniae

Three different mutations were introduced in the polA gene of Streptococcus pneumoniae by chromosomal transformation. One mutant gene encodes a truncated protein that possesses 5' to 3' exonuclease but has lost polymerase activity. This mutation does not affect cell viability. Other mutated forms of polA that encode proteins with only polymerase activity or with no enzymatic activity could not substitute for the wild-type polA gene in the chromosome unless the 5' to 3' exonuclease domain was encoded elsewhere in the chromosome. Thus, it appears that the 5' to 3' exonuclease activity of the DNA polymerase I is essential for cell viability in S. pneumoniae. Absence of the polymerase domain of DNA polymerase I slightly diminished the ability of S. pneumoniae to repair DNA lesions after ultraviolet irradiation. However, the polymerase domain of the pneumococcal DNA polymerase I gave almost complete complementation of the polA5 mutation in Escherichia coli with respect to resistance to ultraviolet irradiation.     

Patch length of localized repair events: role of DNA polymerase I in mutY-dependent mismatch repair.

In vivo experiments with heteroduplex lambda genomes show that the MutY mismatch repair system of Escherichia coli defines an average repair tract that is shorter than 27 nucleotides and longer than 9 nucleotides and extends 3' from the corrected adenine. The phenotype of a mutant defective in DNA polymerase I shows that this enzyme plays a significant, though not an essential, role in the in vivo repair of apurinic sites generated by this system. Evidence is presented that in the absence of polymerase I the repair tracts are modestly longer than in the polA+ extending in the 5' direction from the corrected adenine, suggesting a role for another DNA polymerase.     

Role of deoxyribonucleic acid polymerase III in the repair of single-strand breaks produced in Escherichia coli deoxyribonucleic acid by gamma radiation.

Cell survival, deoxyribonucleic acid (DNA) degradation, and the repair of DNA single-strand breaks were measured for Escherichia coli K-12 pol+, polA1, polC1026(ts), and polA1 polC1026(ts) cells after 137Cs gamma irradiation. The results indicate that DNA polymerase III is required for growth medium-dependent (type III) repair in polA+ or polA cells. In pol+ or polC cells, DNA polymerase I performs type II repair efficiently. The relative deficiencies of each of these strains in DNA repair generally correlate with their relative sensitivities to cell killing and with the extent of DNA degradation observed.     

Genetic and kinetic evidence for different types of postreplication repair in Escherichia coli B.

The changes in molecular weight of deoxyribonucleic acid (DNA) synthesized after ultraviolte irradiation of Escherichia coli WP28 uvrA, and strains additionally mutant at polA, exrA, recA, and exrA and polA loci, were examined by alkaline sucrose gradient centrifugation. In a repari=deficient uvrA recA strain, the frequency of breaks in newly synthesized DNA was equal to that for pyrimidine dimers in parental DNA. Measurements of the amounts and rates of postreplication repair of these breaks indicate that (i) repair is two to three times faster when DNA polymerase I is present, although (ii) almost all breaks are repaired regardless of DNA polymerase I activity. (iii) Increased ultraviolet doses lead to an increase in the proportion of breaks remaining unrepaired in uvrA recA, UVRA exrA, and uvrA exrA polA strains. The numbers of unrepaired breaks resemble the numbers expected if repair of one lesion is prevented by proximity of a second lesion.     

Methyl methane sulfonate-sensitive mutant of Escherichia coli deficient in an endonuclease specific for apurinic sites in deoxyribonucleic acid.

A methyl methane sulfonate (MMS)-sensitive mutant of Escherichia coli AB 1157 was obtained by N-methyl-N'-nitro-N-nitrosoguanidine treatment. The mutant strain, AB 3027, is defective both in endonuclease activity for apurinic sites in deoxyribonucleic acid (DNA) and in DNA polymerase I, as shown by direct enzyme assays. Derivative strains, which retained the deficiency in endonuclease activity for apurinic sties (approximately 10% of the wild-type enzyme level) but had normal DNA polymerase I activity, were obtained by P1-mediated transduction (strain NH5016) or by selection of revertants to decreased MMS sensitivity. These endonuclease-deficient strains are more MMS-sensitive than wild-type strains. Revertants of these deficients strains to normal MMS resistance were isolated. They had increased levels of the endonuclease activity but did not attain wild-type levels. The data suggest that endonuclease for apurinic sites is active in repair of lesions introduced in DNA as a consequence of MMS treatment. Two different endonucleases that specifically attack DNA containing apurinic sites arepresented in E coli K-12. A heat-labile activity, sensitive to inhibition by ethylenediaminetetraacetate, accounts for 90% of the total endonuclease activity for apurinic sties in crude cell extracts. The residual 10% is due to a more heat-resistant activity, refractory to ethylenediaminetetraacetate inhibition. The AB3027 and NH5016 strains have normal amounts of the latter endonuclease but no or very little of the former activity.     

uvrC Gene Function in Excision Repair in Toluene-Treated Escherichia coli

We have examined the role of the uvrC gene in UV excision repair by studying incision, excision, repair synthesis, and DNA strand reformation in Escherichia coli mutants made permeable to nucleoside triphosphates by toluene treatment. After irradiation, incisions occur normally in uvrC cells in the presence of nicotinamide mononucleotide (NMN), a ligase-blocking agent, but cannot be detected otherwise. We conclude that repair incisions are followed by a ligation event in uvrC mutants, masking incision. However, a uvrC polA12 mutant accumulates incisions only slightly less efficiently than a polA12 strain without NMN. Excision of pyrimidine dimers is defective in uvrC mutants (polA(+) or polA12) irrespective of the presence or absence of NMN. DNA polymerase I-dependent, NMN-stimulated repair synthesis, which is demonstrable in wild-type cells, is absent in uvrC polA(+) cells, but the uvrC polA12 mutant exhibits a UV-specific, ATP-dependent repair synthesis like parental polA12 strains. A DNA polymerase I-mediated reformation of high-molecular-weight DNA takes place efficiently in uvrC polA(+) mutants after incision accumulation, and the uvrC polA12 mutant shows more reformation than the polA12 strain after incision. These results indicate that normal incision occurs in uvrC mutants, but there appears to be a defect in the excision of pyrimidine dimers, allowing resealing via ligation at the site of the incision. The lack of NMN-stimulated repair synthesis in uvrC polA(+) cells indicates that incision is not the only requirement for repair synthesis.     

Streptococcus pneumoniae DNA polymerase I lacks 3''-to-5'' exonuclease activity: localization of the 5''-to-3'' exonucleolytic domain.

The Streptococcus pneumoniae polA gene was altered at various positions by deletions and insertions. The polypeptides encoded by these mutant polA genes were identified in S. pneumoniae. Three of them were enzymatically active. One was a fused protein containing the first 11 amino acid residues of gene 10 from coliphage T7 and the carboxyl-terminal two-thirds of pneumococcal DNA polymerase I; it possessed only polymerase activity. The other two enzymatically active proteins, which contained 620 and 351 amino acid residues from the amino terminus, respectively, lacked polymerase activity and showed only exonuclease activity. These two polymerase-deficient proteins and the wild-type protein were hyperproduced in Escherichia coli and purified. In contrast to the DNA polymerase I of Escherichia coli but similar to the corresponding enzyme of Thermus aquaticus, the pneumococcal enzyme appeared to lack 3'-to-5' exonuclease activity. The 5'-to-3' exonuclease domain was located in the amino-terminal region of the wild-type pneumococcal protein. This exonuclease activity excised deoxyribonucleoside 5'-monophosphate from both double- and single-stranded DNAs. It degraded oligonucleotide substrates to a decameric final product.     

Changes in deoxyribonucleic acid polymerase activities in synthesis of deoxyribonucleic acid during sporulation of Bacillus subtilis.

The deoxyribonucleic acid (DNA) polymerase activities in Bacillus subtilis strains Marburg 168 (thy-trp2) and D22, a DNA polymerase I-deficient mutant, were measured at various stages of sporulation. The DNA polymerase I activity, which had decreased after the exponential growth, began to increase at the early stage of sporulation, reached a maximum and then again decreased. The activity of neither DNA polymerase II nor III was observed to change so drastically as that of DNA polymerase I during sporulation. The incorporation of [3H]deoxythymidine 5'-triphosphate ([3H]dTTP) into Brij 58-treated permeable cells increased during sporulation. The stimulation of [3H]dTTP incorporation into the cells by irradiation with ultraviolet light was also observed to coincide with DNA polymerase I activity. In strain D22 the activities of DNA polymerase II and III were almost constant with time. Neither change of [3H]dTTP incorporation into Brij 58-treated cells nor stimulation of incorporation by irradiation with ultraviolet light was observed.     

In vitro characterization of repair synthesis initiated by T4 endonuclease V on a synthetic DNA substrate.

The size of the repair patch produced by E. coli DNA polymerase (Pol I) following the removal of a pyrimidine dimer from DNA in response to the nicking activity of T4 endonuclease (T4 endo V) was determined. A 48-bp DNA containing a pyrimidine dimer at a defined location was labelled in the damaged strand and incubated with T4 endo V and E. coli endonuclease IV. Subsequently, DNA synthesis by DNA Pol I was carried out in the presence of four dNTPs, ATP and DNA ligase. Analysis of the reaction products on a sequencing gel revealed a ladder of only 4-oligonucleotides, 1-4 nucleotides greater in length than the fragment generated by the combined nicking activities of T4 endo V and E. coli endonuclease IV. Thus we conclude that the in vitro repair patch size of T4 endo V is 4 nucleotides and that in some cases the repaired DNA is not ligated.     

In vitro host cell reactivation of alkylated bacteriophage T7 deoxyribonucleic acid by repair-deficient strains of Escherichia coli.

An in vitro system capable of packaging bacteriophage T7 deoxyribonucleic acid (DNA) into phage heads to form viable phage particles has been used to monitor the biological consequences of DNA dam aged by alkylating agents, and an in vitro DNA replication system has been used to examine the ability of alkylated T7 DNA to serve as template for DNA synthesis. The survival of phage resulting from in vitro packaging of DNA preexposed to various concentrations of methyl methane sulfonate or ethyl methane sulfonate closely paralleled the in vivo situation, in which intact phage were exposed to the alkylating agents. Host factors responsible for survival of alkylated T7 have been examined by using wild-type strains of EScherichia coli and mutants deficient in DNA polymerase I (polA) or 3-methyladenine-DNA glycosylase (tag). For both in vivo and in vitro situations, a deficiency in 3-methyladenine-DNA glycosylase dramatically reduced phage survival relative to that in the wild type, whereas a deficiency in DNA polymerase I had an intermediate effect. Furthermore, when the tag mutant was used as an indicator strain, phage survival was enhanced when alkylated DNA was packaged with extracts prepared from a wild-type strain in place of the tag mutant or by complementing a tag extract with an uninfected tag+ extract, indicating in vitro repair during packaging.     

Streptococcus pneumoniae polA gene is expressed in Escherichia coli and can functionally substitute for the E. coli polA gene.

The Streptococcus pneumoniae polA+ gene was introduced into Escherichia coli on the recombinant plasmid pSM31, which is based on the pSC101 replicon. Extracts of E. coli polA5 mutants containing pSM31 showed DNA polymerase activity, indicating that the pneumococcal DNA polymerase I was expressed in the heterospecific host. Complete complementation of the E. coli polA5 mutation by the pneumococcal polA+ gene was detected in excision repair of DNA damage.     

Repair of neocarzinostatin-induced deoxyribonucleic acid damage in human lymphoblastoid cells: possible involvement of apurinic/apyrimidinic sites as intermediates

Neocarzinostatin (NCS) induces repair in a xeroderma pigmentosum lymphoblastoid line deficient in the ability to repair DNA damage induced with (acetoxyacetyl-amino)fluorene. Repair was demonstrated by the induction of repair synthesis and by the disappearance of NCS-induced single-strand breaks and/or alkaline-labile sites in DNA. Estimation of NCS-induced repair patch size, based on the density shift induced in DNA by extensive shear after incubation of treated cells in medium with bromodeoxyuridine or by calculation from the extent of restoration of DNA sedimentation profiles in alkaline sucrose gradients and the amount of repair synthesis measured by the BND cellulose method, indicated that only a few nucleotides were inserted per repaired region. NCS-treated bacteriophage T7 DNA requires incubation with alkaline phosphatase to make it a substrate for DNA polymerase I. NCS-reacted T7 DNA, even after phosphatase treatment, is not a substrate for a DNA polymerase alpha obtained from human lymphoma cells. NCS-treated T7 DNA did serve as a substrate for the DNA polymerase alpha when incubated with an apurinic/apyrimidinic (AP) endonuclease with associated 5'-3'-exonuclease activity. The results suggest that NCS-induced AP sites could be intermediates for the in vivo repair synthesis.     

Generation of single-nucleotide repair patches following excision of uracil residues from DNA.

The extent and location of DNA repair synthesis in a double-stranded oligonucleotide containing a single dUMP residue have been determined. Gently prepared Escherichia coli and mammalian cell extracts were employed for excision repair in vitro. The size of the resynthesized patch was estimated by restriction enzyme analysis of the repaired oligonucleotide. Following enzymatic digestion and denaturing gel electrophoresis, the extent of incorporation of radioactively labeled nucleotides in the vicinity of the lesion was determined by autoradiography. Cell extracts of E. coli and of human cell lines were shown to carry out repair mainly by replacing a single nucleotide. No significant repair replication on the 5' side of the lesion was observed. The data indicate that, after cleavage of the dUMP residue by uracil-DNA glycosylase and incision of the resultant apurinic-apyrimidinic site by an apurinic-apyrimidinic endonuclease activity, the excision step is catalyzed usually by a DNA deoxyribophosphodiesterase rather than by an exonuclease. Gap-filling and ligation complete the repair reaction. Experiments with enzyme inhibitors in mammalian cell extracts suggest that the repair replication step is catalyzed by DNA polymerase beta.     

Escherichia coli uracil- and ethenocytosine-initiated base excision DNA repair: rate-limiting step and patch size distribution

The rate, extent, and DNA synthesis patch size of base excision repair (BER) were measured using Escherichia coli GM31 cell-free extracts and a pGEM (form I) DNA substrate containing a site-specific uracil or ethenocytosine target. The rate of complete BER was stimulated (approximately 3-fold) by adding exogenous E. coli DNA ligase to the cell-free extract, whereas addition of E. coli Ung, Nfo, Fpg, or Pol I did not stimulate BER. Hence, DNA ligation was identified as the rate-limiting step in the E. coli BER pathway. The addition of exogenous DNA polymerase I caused modest inhibition of BER, which was overcome by concomitant addition of DNA ligase. Repair patch size determinations were performed to assess the distribution of DNA synthesis associated with both uracil- and ethenocytosine-initiated BER. During the early phase (0-5 min) of the BER reaction, the large majority of repair events resulted from short patch (1-nucleotide) DNA synthesis. However, during the late phase (>10 min) both short and long (2-20 nucleotide) patches were observed, with long patch BER progressively dominating the repair process. In addition, the patch size distribution was influenced by the ratio of DNA polymerase I to DNA ligase activity in the reaction. A novel mode of BER was identified that involved DNA synthesis tracts of >205 nucleotides in length and termed very-long patch BER. This BER process was dependent upon DNA polymerase I since very-long patch BER was inhibited by DNA polymerase I antibody and addition of excess DNA polymerase I reversed this inhibition.     

In Vitro Packaging of UV Radiation-Damaged DNA from Bacteriophage T7

When DNA from bacteriophage T7 is irradiated with UV light, the efficiency with which this DNA can be packaged in vitro to form viable phage particles is reduced. A comparison between irradiated DNA packaged in vitro and irradiated intact phage particles shows almost identical survival as a function of UV dose when Escherichia coli wild type or polA or uvrA mutants are used as the host. Although uvrA mutants perform less host cell reactivation, the polA strains are identical with wild type in their ability to support the growth of irradiated T7 phage or irradiated T7 DNA packaged in vitro into complete phage. An examination of in vitro repair performed by extracts of T7-infected E.coli suggests that T7 DNA polymerase may substitute for E. coli DNA polymerase I in the resynthesis step of excision repair. Also tested was the ability of a similar in vitro repair system that used extracts from uninfected cells to restore biological activity of irradiated DNA. When T7 DNA damaged by UV irradiation was treated with an endonuclease from Micrococcus luteus that is specific for pyrimidine dimers and then was incubated with an extract of uninfected E. coli capable of removing pyrimidine dimers and restoring the DNA of its original (whole genome size) molecular weight, this DNA showed a higher packaging efficiency than untreated DNA, thus demonstrating that the in vitro repair system partially restored the biological activity of UV-damaged DNA.     

Repair resynthesis in Escherichia coli mutants deficient in single-stranded DNA-binding protein

A series of Escherichia coli strains deficient in single-stranded DNA-binding protein (SSB) and DNA polymerase I was constructed in order to analyze the effects of these mutations on DNA repair resynthesis after UV-irradiation. Since SSB has been suggested to play a role in protecting single-stranded regions which may transiently exist during excision repair and since long single-stranded regions are believed to occur frequently as repair intermediates in strains deficient in DNA polymerase I, studies of repair resynthesis and strand rejoining were performed on strains containing both the ssb-1 and polA1 mutations. Repair resynthesis appears to be slightly decreased in the ssb-1 strain at 42 degrees C relative to the wild-type; however, this effect is not enhanced in a polA1 derivative of this strain. After UV-irradiation, the single-strand molecular weight of the DNA of an ssb-1 strain decreases and fails to recover to normal size. These results are discussed in the context of long patch repair as an inducible component of repair resynthesis and of the protection of intermediates in the excision repair process by SSB. A direct role for SSB in repair resynthesis involving modulation of the proteins involved in this mode of DNA synthesis (particularly stimulation of DNA polymerase II) is not supported by our findings.     

Down-regulation of DNA repair synthesis at DNA single-strand interruptions in poly(ADP-ribose) polymerase-1 deficient murine cell extracts

The functional involvement of poly(ADP-ribose) polymerase-1 (PARP-1) in the repair of DNA single- and double-strand breaks, DNA base damage, and related repair substrate intermediates remains unclear. Using an in vitro DNA repair assay and cell extracts derived from PARP-1 deficient or wild-type murine embryonic fibroblasts, we investigated the DNA synthesis and ligation steps associated with the rejoining of DNA single-strand interruptions containing 3'-OH, and either 5'-OH or 5'-P termini. Complete repair leading to DNA rejoining was similar between PARP-1 deficient cells and wild-type controls and poly(ADP-ribose) synthesis was, as expected, greatly reduced in PARP-1 deficient cell extracts. The incorporation of [32P]dCMP into repaired DNA at the site of a lesion was reduced two-three-fold in PARP-1 deficient cell extracts, demonstrating a decrease in repair patch size. Addition of purified PARP-1 to levels approximating those present in wild-type extracts did not stimulate DNA repair synthesis. We conclude that PARP-1 is not required for the efficient processing and rejoining of single-strand interruptions with defined 3'-OH and 5'-OH or 5'-P termini. Decreased DNA repair synthesis observed in PARP-1 deficient cell extracts is associated with reduced cellular expression of several factors required for long-patch base excision repair (BER), including FEN-1 and DNA ligase I.     

Temperature-Sensitive Mutants for the Replication of Plasmids in Escherichia coli: Requirement for Deoxyribonucleic Acid Polymerase I in the Replication of the Plasmid ColE1

An Escherichia coli mutant (polA1), defective in deoxyribonucleic acid (DNA) polymerase I, (EC 2.7.7.7) is unable to maintain colicinogenic factor E1 (ColE1), whereas several sex factor plasmids are maintained normally in this strain. polA1 mutant strains containing these sex factor plasmids do not exhibit a readily detectable plasmid-induced polymerase activity. A series of E. coli mutants that are temperature sensitive for ColE1 maintenance, but able to maintain other plasmids, were isolated and shown to fall into two phenotypic groups. Mutants in one group are defective specifically in ColE1 maintenance at 43 C, but exhibit normal DNA polymerase I activity. Mutations in the second group map in the polA gene of E. coli, and bacteria carrying these mutations are sensitive to methylmethanesulfonate (MMS). Revertants that were selected either for MMS resistance or the ability to maintain ColE1 were normal for both properties. The DNA polymerase I enzyme of two of these mutants shows a pronounced temperature sensitivity when compared to the wild-type enzyme. An examination of the role of DNA polymerase I in ColE1 maintenance indicates that it is essential for normal replication of the plasmid. In addition, the presence of a functional DNA polymerase I in both the donor and recipient cell is required for the ColV-promoted conjugal transfer of ColE1 and establishment of the plasmid in the recipient cell.