Accumulating evidence indicates that there are at least two distinct modes of DNA binding by Rep68/78, corresponding to the multiple roles that these proteins play during viral replication. One of the initial functions of Rep68/78 is to recognize and bind specifically to the viral origin of replication. This is mediated by the N-terminal endonuclease domain, which binds specifically to the double-stranded form of the RBS, most likely forming a spiral of Rep molecules along the DNA, as seen for the isolated endonuclease domain (21
). This type of spiral assembly presumably accounts for the observation of AAV2 Rep68/78 hexamers in the presence of AAV origin sequences (10
), although it has recently been suggested that this complex might be only pentameric (34
). The second mode of binding occurs in the presence of random ssDNA or dsDNA and relates to the helicase function of Rep68/78. These assemblies seem likely to be hexameric or dodecameric, although double octamers have been reported (34
studies to understand the apparent ability of Rep68/78 to switch conformations from a spiral assembly to a planar ring expected for an SF3 helicase—or to be capable of adopting both conformations—have been hampered by the poor biophysical properties of the AAV2 and AAV5 Rep 68/78 proteins, particularly in the low-ionic-strength buffer conditions required to detect DNA binding (10
; this study). Here, we circumvented this problem by creating a series of deletion versions of Rep68/78 in an effort to understand the contributions of various domains to DNA binding and helicase assembly. Our results establish that the 23-amino-acid linker sequence located between the endonuclease and helicase domains of AAV5 Rep68/78 proteins is a crucial contributor to the ability of the AAV5 Rep helicase domain to form discrete oligomeric complexes on both ssDNA and dsDNA. These complexes are functionally active, demonstrating both stimulated ATPase and helicase activity relative to the isolated helicase domain.
The linker region appears to be a strong driver of DNA binding. For example, it confers nonspecific dsDNA binding to the isolated helicase domain (), the endonuclease domain (C), and even the small subdomain comprised of only the four-helix bundle that precedes the Rep AAA+ domain (A). However, it is possible that the architecture of these protein-DNA complexes may be fundamentally different. For example, in the case of Rep198-275, which does not have the AAA+ domain, DNA binding is evident on short oligonucleotides, and it appears that the number of bound protein molecules depends on the DNA length. In contrast, Rep198-489 forms discrete hexameric complexes () only on DNA longer than ~30 nucleotides. Thus, although the AAA+ domain does not detectably contribute to DNA binding, it is clearly important in establishing the architecture of the assembled proteins on DNA. Stable assembly on DNA appears to require sufficient DNA to fully pass through the central cavity of the assembled hexamer, since 30 nucleotides is on the same order as the number of base pairs of dsDNA that might be expected to be accommodated in the observed central channels of the SF3 helicase domains that have been structurally characterized. In the case of the SV40 helicase domain, the central channel is ~80 Å long (30
), whereas that of the E1 helicase domain is ~60 Å (12
Our data strongly suggest that the linker region plays an important role in the oligomerization of the larger Rep proteins on nonspecific ssDNA and dsDNA. It is tempting to speculate that the linker might serve as a hook that helps to hold Rep68/78 in place on DNA as it transitions between the first mode and second mode of DNA binding. During the transition, the linker region might be engaged as we observed for Rep198-275, perhaps forming a “spiral coat” along the DNA molecule limited only by the availability of accessible DNA or the number of Rep protomers and recapitulating the spiral observed for endonuclease binding to the RBS (21
). In the second mode of binding, the presence of the linker region stimulates both the ATPase and helicase activities, suggesting that it actively contributes to the organization of the assembly.
The importance of the linker region has been previously demonstrated for AAV2 since mutation of either R217 or K219 (corresponding to K213 and K215 of AAV5; underlined in A) to alanine in Rep78 results in a protein unable to nick at the trs or mediate site-specific integration into AAVS1, whereas RBS binding is maintained (46
). The lost activities are consistent with a model in which mutations in the linker region prevent the formation of an active helicase-competent assembly, shown to be necessary for trs nicking (5
). Each of our cluster mutants 1 to 4 contained a mutation of either K213 or K215, and the associated loss of DNA binding ( and ) provides a potential mechanistic explanation for the observations of Urabe et al. (46
For other members of the SF3 helicase superfamily, such as T-Ag and E1, combined biochemical and structural studies have shown that the N-terminal origin binding domain and the helicase AAA+ domain are linked by an intervening domain that mediates oligomerization (12
; reviewed in reference 22
). In the case of T-Ag, the intervening domain is a Zn2+
-binding domain; for E1, the intervening domain forms a four-helix bundle that is structurally unrelated to the T-Ag Zn2+
-binding domain but is in turn structurally homologous to the hexamerization domain of the RCR replication initiator protein of pMV158 (3
). AAV Rep appears to have dealt with the intervening region somewhat differently by dividing it into two parts depending on protein context. For Rep40 and Rep52, the intervening region consists of a small four-helix bundle that alone does not have potent DNA binding or oligomerization properties. For the larger Rep proteins, the intervening domain appears to be functionally comprised of the four-helix bundle supplemented by the linker.
Since Rep40 and Rep52 lack the linker sequence, our results here do not shed direct light onto the functions of these smaller Rep proteins during genome packaging, which might be reasonably expected to be mediated by a multimeric molecular motor that pumps DNA into preformed capsids. It would be interesting to establish whether the ability of Rep40 to oligomerize when extended by a short peptide sequence mimics the properties of Rep40 and Rep52 when bound to other proteins or protein complexes such as the viral capsid proteins (1
). The need for accessory proteins to aid the assembly of an active motor protein is not unprecedented: for example, the eukaryotic MCM2-7 replicative helicase requires the assistance of accessory proteins ORC1-6 and Cdc6 to load onto DNA (2
), and deposition of the E. coli
DnaB helicase at replication forks requires direct interactions with two other proteins, DnaA and DnaC (25
Hexameric helicases continue to intrigue, and the relevance of double hexamers encircling dsDNA continues to be discussed (4
). We do not yet know whether the double hexamers we observed represent an authentic assembly along the AAV replication pathway. It is nonetheless clear that the linker region is not a passive tether joining two independent protein domains but rather contributes to the oligomeric properties of AAV Rep in the presence of DNA.