The requirement for both DNA polymerase and 5'' to 3'' exonuclease activities of DNA polymerase I during Tn5 transposition

Summary By assaying transposition of Tn5 from b221 cI857 rex::Tn5 (Berg 1977) in PolA-proficient and deficient cells, both the polymerase activity and 5 to 3 exonuclease acivity of DNA polymerase I have been shown to be required for transposition. This requirement could not be observed in three other systems in which the transposon donor replicon had existed in the PolA-proficient and deficient cells before the transposition event to be assayed occurred. By analogy to Tn3, this may indicate that the repressor encoded by Tn5 has already been expressed and hence become rate-limiting in the overall transposition process, even in PolA-deficient cells still possessing a residual activity. One polA mutant was found among more than 50 transposition-deficient (tnp) mutants isolated by the use of b221 cI857 rex::Tn5.     

Alteration of the exonuclease activities of DNA polymerase I by captan

DNA polymerase I is a multifaceted enzyme with one polymerizing and two exonuclease activities. Captan was previously shown to be an inhibitor of this enzyme's polymerizing activity and this report measures the effects of captan on the two exonuclease activities. When the holoenzyme was tested, captan enhanced the degradation of poly(dA-dT), T7 DNA and, to a significantly lesser extent, heat-denatured DNA. However, when the effects of captan were tested as a function of substrate concentration, the stimulatory influence was measured only at high substrate concentrations. At low concentrations of DNA, captan was inhibitory. Inhibition and enhancement each showed an ED50 of the same value (approx. 100 microM). By assaying the two exonuclease activities separately it was shown that the differential effect on the holoenzyme by captan was the result of a combined inhibition of the 3'----5' exonuclease and enhancement of the 5'----3' exonuclease. Klenow fragment with poly(dA-dT) as substrate was used to assay for 3'----5' exonuclease activity. Captan inhibited this exonuclease and the inhibition could be prevented by the addition of greater concentrations of substrate. Holoenzyme and poly(rA)-poly(dT) were used to assay for 5'----3' exonucleolysis, which was enhanced at higher concentrations of substrate in the presence of captan.     

Deoxyribonucleic acid repair in Escherichia coli mutants deficient in the 5''----3'' exonuclease activity of deoxyribonucleic acid polymerase I and exonuclease VII.

A series of Escherichia coli strains deficient in the 5'----3' exonuclease activity associated with deoxyribonucleic acid (DNA) polymerase I (exonuclease VI) and exonuclease VII has been constructed. Both of these enzymes are capable of pyrimidine dimer excision in vitro. These strains were examined for conditional lethality, sensitivity to ultraviolet (UV) and X-irradiation, postirradiation DNA degradation, and ability to excise pyrimidine dimers. It was found that strains deficient in both exonuclease VI (polAex-) and exonuclease VII (xseA-) are significantly reduced in their ability to survive incubation at elevated temperature (43 degrees C) beyond the reduction previously observed for the polAex single mutants. The UV and X-ray sensitivity of the exonuclease VI-deficient strains was not increased by the addition of the xseA7 mutation. Mutants deficient in both enzymes are about as efficient as wild-type strains at excising dimers produced by up to 40 J/m2 UV. At higher doses strains containing only polAex- mutations show reduced ability to excise dimers; however, the interpretation of dimer excision data at these doses is complicated by extreme postirradiation DNA degradation in these strains. The additional deficiency in the polAex xseA7 double-mutant strains has no significant effect on either postirradiation DNA degradation or the apparent deficiency in dimer excision at high UV doses observed in polAex single mutants.     

Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I

Circular (e.g. simian virus 40) and linear (e.g. λ phage) DNAs have been labeled to high specific radioactivities (>10⁸ cts/min per μg) in vitro using deoxynucleoside [α-³²P]triphosphates (100 to 250 Ci/mmol) as substrates and the nick translation activity of Escherichia coli DNA polymerase I. The reaction product yields single-stranded fragments about 400 nucleotides long following denaturation. Because restriction fragments derived from different regions of the nick-translated DNA have nearly the same specific radioactivity (cts/min per 10[su3] bases), we infer that nicks are introduced, and nick translation is initiated, with equal probability within all internal regions of the DNA. Such labeled DNAs (and restriction endonuclease fragments derived from them) are useful probes for detecting rare homologous sequences by in situ hybridization and reassociation kinetic analysis.     

Localization of the exonuclease and polymerase domains of Bacillus subtilis DNA polymerase III.

Structural gene mutants were cloned and exploited to identify the major catalytic domains of Bacillus subtilis DNA polymerase III (BsPolIII), a 162.4-kDa [1437 amino acids (aa)] polymerase: 3'-5' exonuclease (Exo) required for replicative DNA synthesis. Analysis of the sequence, mutagenicity, and catalytic behavior of natural and site-directed point mutants of BsPolIII unequivocally located the domain involved in exonuclease catalysis within a 155-aa residue segment displaying homology with the Exo domain of Escherichia coli DNA polymerase I. Sequence analysis of four structural gene mutations which specifically alter then enzyme's reactivity to the inhibitory dGTP analog, 6-(p-hydroxyphenylhydrazino)uracil, and the inhibitory arabinonucleotide, araCTP, defined a domain (Pol) involved in dNTP binding. The Pol domain was in the C-terminal fourth of the enzyme within a 98-aa segment spanning aa 1175-1273. The primary structure of the domain was unique, displaying no obvious conservation in any other DNA polymerase, including the distantly related PolIIIs of the Gram- organisms, E. coli and Salmonella typhimurium.     

Isolation of a yeast single-strand deoxyribonucleic acid binding protein that specifically stimulates yeast DNA polymerase I

We sought a protein from yeast that would bind more strongly to single-stranded DNA than to duplex DNA and would stimulate the activity of the major yeast DNA polymerase, but not polymerases from other organisms. We isolated a protein that binds about 200 times more strongly to single-stranded DNA than duplex DNA and stimulates yeast DNA polymerase I activity 4-5-fold. It inhibits synthesis catalyzed by calf thymus DNA polymerase alpha and has little effect on T4 DNA polymerase. This yeast protein, SSB-1, has a molecular weight of approximately 40 000. At apparent saturation there is one protein molecule bound per 40 nucleotides. Protein binding causes the single-stranded DNA molecule to assume a relatively extended conformation. It binds to single-stranded RNA as strongly as to DNA. SSB-1 increases the initial rate of polymerization catalyzed by yeast DNA polymerase I apparently by increasing the processivity of the enzyme. We estimate there are 7500-30 000 molecules of SSB-1 per yeast cell, enough to bind at least 400-1600 nucleotides per replication fork. Thus it is present in sufficient abundance to participate in DNA replication in vivo in the manner suggested by these in vitro experiments.     

Effects of vinylphosphonate internucleotide linkages on the cleavage specificity of exonuclease III and on the activity of DNA polymerase I

We have previously reported the synthesis of vinylphosphonate-linked thymidine dimers and their incorporation into synthetic oligonucleotides to create vinylphosphonate internucleotide linkages in the DNA. Such linkages have a profound effect on DNA backbone rotational flexibility, and we have shown that the PcrA helicase, which requires such flexibility, is inhibited when it encounters these linkages on the translocating strand. In this study, we have investigated the effects of these linkages on the dsDNA specific exonuclease III and on the ssDNA specific mung bean nuclease to establish whether our modification confers resistance to nucleases making it suitable for antisense therapy applications. We also investigated the effect on DNA polymerase I to establish whether we could in the future use this enzyme to incorporate these linkages in the DNA. Our results show that a single modification does not affect the activity of DNA polymerase I, but four vinylphosphonate linkages in tandem inhibit its activity. Furthermore, such linkages do not confer significant nuclease resistance to either exonuclease III or mung bean nuclease, but unexpectedly, they alter the cleavage specificity of exonuclease III.     

DNA polymerase III requirement for repair of DNA damage caused by methyl methanesulfonate and hydrogen peroxide.

The pcbA1 mutation allows DNA replication dependent on DNA polymerase I at the restrictive temperature in polC(Ts) strains. Cells which carry pcbA1, a functional DNA polymerase I, and a temperature-sensitive DNA polymerase III gene were used to study the role of DNA polymerase III in DNA repair. At the restrictive temperature for DNA polymerase III, these strains were more sensitive to the alkylating agent methyl methanesulfonate (MMS) and hydrogen peroxide than normal cells. The same strains showed no increase in sensitivity to bleomycin, UV light, or psoralen at the restrictive temperature. The sensitivity of these strains to MMS and hydrogen peroxide was not due to the pcbAl allele, and normal sensitivity was restored by the introduction of a chromosomal or cloned DNA polymerase III gene, verifying that the sensitivity was due to loss of DNA polymerase III alpha-subunit activity. A functional DNA polymerase III is required for the reformation of high-molecular-weight DNA after treatment of cells with MMS or hydrogen peroxide, as demonstrated by alkaline sucrose sedimentation results. Thus, it appears that a functional DNA polymerase III is required for the optimal repair of DNA damage by MMS or hydrogen peroxide.     

Periodicity of exonuclease III digestion of chromatin and the pitch of deoxyribonucleic acid on the nucleosome

Exonuclease III has previously been shown to pause about every 10 nucleotides along the 3' strands while it invades the nucleosome core. Here, the exact periodicity of this digestion, i.e., the spacing of the pauses, was determined. Results showed that the exonuclease digests the first 20 nucleotides at the edge of the nucleosome core with a periodicity of approximately 11 nucleotides; in contrast, DNA closer to the center of the particle is digested with a smaller periodicity of about 10 nucleotides. These figures differ from the known periodicity of DNase I digestion, approximately 10 and 10.5 nucleotides at the edge and in the center of the nucleosome, respectively. Moreover, as shown by sedimentations in sucrose gradients, the structure of the nucleosome does not appear to be significantly altered by the gradual destruction of its DNA moiety by the exonuclease. Such stability of the nucleosome, along with other complementary observations, indicates that the transition in the digestion periodicity of the exonuclease may not be the consequence of a structural rearrangement of the particle upon trimming. This transition may rather be ascribed to the properties of the native nucleosome and to the intrinsic mechanism of action of the enzyme. Finally, evidence is presented which suggests that the exonuclease 10-nucleotide periodicity of digestion of the inner region of the nucleosome reflects a 10 base pair/turn pitch of the DNA in that region.