Mapping target site selection for the non-specific nuclease activities of retroviral integrase.
To identify the parts of retroviral integrase that interact with its DNA substrates, we compared the patterns of target site usage by chimeric enzymes and protein fragments in assays that reveal integrase's non-specific nuclease activities. The central region of 12 chimeric proteins between the human immunodeficiency virus type 1 and visna virus integrases was found to be responsible for selecting non-viral target DNA sites when small alcohols provide the attacking nucleophilic OH group during non-specific alcoholysis assays. Testing deletion derivatives of the integrase protein in this assay, which has similarities to the DNA joining reaction that occurs during retroviral integration, defined a smaller central domain that is sufficient for activity. Thus, this core domain likely contains both the host DNA site and the nucleophile site. Surprisingly, the region of integrase responsible for selecting non-viral target DNA sites when the viral DNA end is the attacking nucleophile could not similarly be mapped with the standard oligonucleotide joining assay. We therefore tested the proteins in a more sensitive assay that displays preferred sites of viral DNA insertion in a plasmid DNA target. All 12 chimeras yielded novel patterns compared with the wild-type enzymes in this assay, although local insertion patterns indicated that the central domain plays an important role in target site selection. Together, these data suggest that other protein regions must be involved when the attacking nucleophilic group is provided by viral DNA. Because specific recognition of viral DNA ends was previously mapped to the central domain, two different regions of integrase must interact with retroviral DNA.
Katzman
M
M
Department of Medicine, Section of Infectious Diseases, Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, 500 University Drive, Mail Services H036, Hershey, PA 17033-2390, USA. mkatzman@psu.edu
Sudol
M
M
Pufnock
J S
JS
Zeto
S
S
Skinner
L M
LM
D000438:Alcohols / Q000378:metabolism; D002384:Catalysis; D004247:DNA / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism*; D019427:HIV Integrase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics; D006801:Humans; D019426:Integrases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016662:Virus Integration; D014790:Visna-maedi virus / Q000201:enzymology* / Q000235:genetics
D000438:Alcohols; D004279:DNA, Viral; D011993:Recombinant Fusion Proteins; D004247:DNA; D019427:HIV Integrase; D019426:Integrases; D003851:Deoxyribonucleases