The amino acid linker between the endonuclease and helicase domains of adeno-associated virus type 5 Rep plays a critical role in DNA-dependent oligomerization.
The adeno-associated virus (AAV) genome encodes four Rep proteins, all of which contain an SF3 helicase domain. The larger Rep proteins, Rep78 and Rep68, are required for viral replication, whereas Rep40 and Rep52 are needed to package AAV genomes into preformed capsids; these smaller proteins are missing the site-specific DNA-binding and endonuclease domain found in Rep68/78. Other viral SF3 helicases, such as the simian virus 40 large T antigen and the papillomavirus E1 protein, are active as hexameric assemblies. However, Rep40 and Rep52 have not been observed to form stable oligomers on their own or with DNA, suggesting that important determinants of helicase multimerization lie outside the helicase domain. Here, we report that when the 23-residue linker that connects the endonuclease and helicase domains is appended to the adeno-associated virus type 5 (AAV5) helicase domain, the resulting protein forms discrete complexes on DNA consistent with single or double hexamers. The formation of these complexes does not require the Rep binding site sequence, nor is it nucleotide dependent. These complexes have stimulated ATPase and helicase activities relative to the helicase domain alone, indicating that they are catalytically relevant, a result supported by negative-stain electron microscopy images of hexameric rings. Similarly, the addition of the linker region to the AAV5 Rep endonuclease domain also confers on it the ability to bind and multimerize on nonspecific double-stranded DNA. We conclude that the linker is likely a key contributor to Rep68/78 DNA-dependent oligomerization and may play an important role in mediating Rep68/78's conversion from site-specific DNA binding to nonspecific DNA unwinding.
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Jenna E
JE
Maggin
Jeffrey A
JA
James
Joshua S
JS
Chappie
Fred
F
Dyda
Alison Burgess
AB
Hickman
D020816:Amino Acid Motifs; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000229:Dependovirus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D004279:DNA, Viral; D014764:Viral Proteins; D004720:Endonucleases; D004265:DNA Helicases