Methods have been developed for the comprehensive analysis of a gene by construction of a saturating or near-saturating library of mutants (5,78,83). This approach has defined domain boundaries, provided functional maps, and given insights into previously predicted unstructured loops (4,5,50,71,73,78,83). In this report, this method of insertional functional mapping is applied to three catalytically related domains: the Moloney murine leukemia virus (M-MuLV) RNase H domain of the reverse transcriptase (RT), and the M-MuLV and human immunodeficiency virus type 1 (HIV-1) integrase (IN) proteins. Inclusion of the HIV-1 IN protein assisted comparison and model building, since structural information is available (7,18,26,34,35,37,87). In the retroviral life cycle, the RNase H activity is required for viral replication during the conversion of the viral RNA (vRNA) into double-stranded DNA through the RNA-DNA intermediate. The IN protein is required for the insertion of the double-stranded DNA into the host chromosome, establishing the integrated provirus.The replication and integration of retroviral particles are two distinct yet interrelated processes. Replicative complexes and preintegrative complexes have been purified and characterized from infected cells (6, 9, 17, 28-30, 39, 52, 53, 55, 56, 66, 67). Within viral species as well as between viral species, the composition of replicative complexes differs from that of preintegrative complexes. Interactions between RT and IN are also reported (40,69,89,90,96), and multiple mutations of IN are known to alter viral replication (27,(58)(59)(60). Despite extensive efforts, the assembly of these complexes is not well understood. These studies have been assisted by structural studies. A structure of the M-MuLV RT has recently been reported (21), as have structures of related retroviral IN subdomains (8,14,18,19,26,35,43,87,94). However, to date, neither a structure of a complete retroviral IN protein nor one of a subdomain in complex with DNA has been obtained.The ability of retroviral particles to stably integrate into the host genome is a great benefit for gene delivery, but the potential for insertional mutagenesis cannot be overlooked (15,22,38,63). Schemes to target integration into alternative positions within the host chromosome to avoid this issue frequently involve generation of fusion proteins with novel targeting domains (10,48,84). The linker insertion genetic footprint provides a means to identify nonessential regions