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Hydroxyurea (HU) has been used successfully (Warner el al. , 1970; Hercules et al. , 1971) to select bacteriophage T4 mutants which are deficient in degrading Escherichia coli DNA after infection. Using a different set of plating conditions, HU has also been used to isolate another set of T4 mutants ( hus ) ( Goscin and Hall, 1972 ). All the backcrossed hus mutants degrade bacterial DNA normally. The original hus 13 contained another mutation N. The double mutant hus 13-N degrades E. coli DNA slowly in the absence of phage DNA synthesis, leaving DNA fragments with a molecular weight midway between the sizes of fragments left by mutants defective in genes 46 and 47 and by endonuclease II ( den A) mutants at similar times after infection. Endonuclease IV activity is normal in hus 13-N infected cells. The HU sensitivity of three mutants, hus 3 (which is deficient in exonuclease A), hus 7 and hus 23 is partially due to inefficient utilization of host-derived deoxyribonucleotides as substrates for phage DNA synthesis. This is related to the DNA delay phenotype of these 3 mutants. The mutants hus 7 and hus 23 also appear to package phage DNA into phage particles inefficiently. The backcrossed hus 13 is apparently HU sensitive due to a defect in packaging DNA into phage particles. The HU sensitivity of two other mutants ( hus 19 and hus 20) remains unexplained as these mutants degrade bacterial DNA normally, synthesize phage DNA normally using host-derived deoxyribonucleotides, and package the DNA into phage particles efficiently.

DNA polymerase delta from rabbit bone marrow has an associated 3'-5'-exonuclease. Previous studies demonstrated a Stokes radius of 45.5 A by gel filtration and a sedimentation coefficient of 6.5 S by zone sedimentation. Thus, a molecular weight of 122000 and a frictional coefficient of 1.39 were calculated [Byrnes, J. J., & Black, V. L. (1978) Biochemistry 17, 4226-4231]. Several problems obstructed further purification and definition of DNA polymerase delta. The small amount of protein obtained limited further purification as the nonspecific loss of enzyme in subsequent procedures was excessive. Furthermore, the amount of protein recovered was insufficient for conventional analysis. These difficulties have been overcome, and DNA polymerase delta has been purified to apparent homogeneity. Under conditions of nondenaturing microgel electrophoresis, DNA polymerase b aggregates to molecular weight species of 300000 and higher. In situ assays for DNA polymerase and exonuclease in these gels generate concordant activity profiles. Upon sodium dodecyl sulfate gel electrophoresis, delta is a single polypeptide of 122000 apparent molecular weight. The DNA polymerase incorporates between 250000 and 300000 nmol of thymidine deoxyribonucleoside monophosphate (dTMP) into poly(dA)/oligo(dT) (mg of protein)-1 h-2 at 37 degrees C; the exonuclease simultaneously hydrolyzes 13% of the newly synthesized DNA. Aphidicolin, considered to be a specific inhibitor of DNA polymerase alpha, inhibits both the DNA polymerase and 3'-5'-exonuclease activities of delta. DNA polymerase alpha from rabbit bone marrow does not share a common subunit with delta. Therefore, aphidicolin binding is not specific for alpha, and conclusions based upon the supposition that it is must be reconsidered.

Modification of the purification procedures for rabbit bone marrow DNA polymerase [Byrnes, J.J., & Black, V.L. (1978) Biochemistry 17, 4226-4231] has increased the yield and stability of the enzyme thus allowing further purification. In particular, the higher molecular weight form, alpha 1, has been more abundant. Additional purification has been obtained upon phosphocellulose and chromatofocusing column chromatography. SDS slab gel electrophoretic analyses of the eluates demonstrate a 135,000 molecular weight polypeptide in nearly pure form which correlates with DNA polymerase activity. Approximately 200,000 nmol of thymidine monophosphate is incorporated into DNA (mg of protein) -1h -1 at 37 degrees C. Similar to DNA polymerase alpha from other sources this enzyme is an acidic protein, is very sensitive to aphidicolin, and has no detectable 3' to 5' nuculease activity.