Nucleic Acids Research

T7 gene 6 exonuclease has been shown to have an RNase H activity as well as a double-strand specific DNase activity by the following experiments: The RNase H activity coelutes with the DNase activity from DEAE-cellulose, phosphocellulose, hydroxyapatite, and Sephadex G-200 columns. Gene 6 exonuclease specified by a T7 strain with a temperature sensitive mutation in gene 6 has an extremely heat-labile RNase H activity as well as a heat-labile DNase activity. T7 gene 6 exonuclease degrades the RNA region of a poly(A)-poly(dT) hybrid polymer exonucleolytically from the 5' terminus, releasing a rlbonucleoside 5'-monophosphate product. When the RNA strand of a X174 RNA-DNA hybrid molecule synthesized with 15. coli RNA polymerase is degraded, a ribonucleoside triphosphate is produced from the 5'-triphosphate terminus. Participation of T7 gene 6 exonuclease in the removal of primer RNA in discontinuous replication of T7 DNA is discussed. INTRODUCTION Analyses performed in this laboratory of nascent short DNA in various prokaryotic systems have shown that RNA is linked to the 5' termini of the pieces (1, 2, 3, 4, 5, 6). This supports the assumption made earlier that synthesis of DNA pieces in discontinuous replication is primed by RNA that is removed prior to the joining of DNA pieces (7). Presence of RNA-linked DNA pieces has been also reported from other groups (8). In I!_. coli, degradation of primer RNA may result from the nick translation activity of DNA polymerase I (9, 10, 11), because RNA-linked nascent short DNA accumulate when the 5'->3' exonuclease activity or/and polymerase activity of the enzyme are deficient (1, 2, 3). In support of this; DNA polymerase I degrades the RNA of RNA-DNA hybrids in vitro, i. e. it has RNase H activity (12, 13, 14). Our previous study of T7 phage infected IJ. coli showed that RNA-linked nascent T7 DNA accumulates in the absence of functional T7 gene 6 (6). The accumulation is particularly pronounced when both gene 6 exonuclease and host DNA polymerase I are deficient, even though the deficiency of the latter © Information Retrieval Limited 1 Falconberg Court London W1V5FG England 4245 Nucleic Acids Research enzyme alone does not cause this accumulation. T7 gene 6 codes for a 5'-»3 exonuclease that is specific for double-stranded DNA (15, 16). Because our results indicate that it removes T7 primer RNA, we have tested if it also has RNase H activity. We find that purified gene 6 protein indeed degrades RNA in variety of RNA-DNA hybrid molecules. The exonucleolytic degradation from the 5 terminus releases ribonucleoside 5' monophosphates. The enzyme also digests 5'triphosphate terminated RNA which is hydrogen bonded to DNA.