A LARGE DELETION IN THE CONNECTION SUBDOMAIN OF MURINE LEUKEMIA-VIRUSREVERSE-TRANSCRIPTASE OR REPLACEMENT OF THE RNASE-H DOMAIN WITH ESCHERICHIA-COLI RNASE-H RESULTS IN ALTERED POLYMERASE AND RNASE-H ACTIVITIES

The functional relationship between the polymerase and RNase H domains of reverse transcriptase (RT) was investigated by studying the activi ties of AKR murine leukemia virus (MuLV) enzymes. In addition to the w ild type, an RNase H-minus RT missing the entire RNase H domain and tw o other mutants having abnormal polymerase:RNase H ratios were express ed. These mutants include (i) a chimeric protein in which the MuLV RNa se H domain was replaced by the entire Escherichia coli RNase H sequen ce and (ii) an RT with a 126 amino acid deletion in a region analogous to the ''connection'' subdomain in the p66 subunit of human immunodef iciency virus type 1 RT (Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A., & Steitz, T. A. (1992) Science 256, 1783-1790). With the wild-type RT, the major RNase H cleavage reaction was coordinated wit h DNA synthesis and occurred at a position corresponding to 15 nucleot ides from the 3'-terminus of the DNA primer. Additional cleavages clos er to the 5'-end of the RNA were explained in terms of a model relatin g binding of the RNA.DNA hybrid substrate and enzyme structure. The ch imeric RT behaved like E. coli RNase H, exhibited 300-fold higher RNas e H activity than wild-type RT, and was limited in its ability to synt hesize DNA. Qualitative and quantitative changes in the polymerase and RNase H activities of the deletion mutant were also observed. The RNa se H domain appeared to function independently of the polymerase domai n, supporting the idea that the proper spatial relationship between th e two active centers was disrupted by the mutation. Taken together, ou r results indicate that alteration of the normal polymerase:RNase H ra tio can have profound effects on both polymerase and RNase H cleavage activities, as expected for an enzyme with two interdependent domains.