Part:BBa_K404001

[AAV2]-Rep-VP123
Rep78
Regulated by the p5 promoter, Rep78 is the largest non-structural protein found in the wtAAV. Besides regulation of gene expression and viral genome replication, Rep78 has been found to play a functional role in AAV site-specific integration into the human genome (Hüser et al., 2010). In absence of Ad helper viruses, overexpression of Rep78 leads to cell cycle arrest by interacting with cell-cycle regulating phosphatases causing DNA damage by its intrinsic endonuclease activity (Berthet, Raj, Saudan, & Beard, 2005) and induces apoptosis. Due to its ability to bind to the Rep binding site (RBS) in the p5 integration-efficient element (p5IEE) of the p5 promoter, Rep78 mediates gene expression and retain a constant level of Rep proteins by suppressing transcriptional activity of the p5 promoter in absence of Ad viruses (Yue et al., 2010). Interaction of Rep78 with cellular factors such as transcription factors (Lackner & Muzyczka, 2002) provides the basis for gene regulation by Rep78 in associated with endogenous molecules.
Rep68
Rep68 is a regulatory protein driven by the p5 promoter with an apparent molecular weight of 68 kDa lacking 92 amino acids from the carboxy terminus due to splicing of mRNA coding for the two larger Rep proteins. The non-structural protein Rep68 belongs to the superfamily 3 (SF3) helicase found in other small DNA and RNA viruses such as simian virus 40 (SV40) and bovine papillomavirus (Mansilla-Soto et al., 2009). Formation of oligomeric complexes of Rep proteins provides the basis for the functional versatility of the two larger regulatory proteins. The AAA+ motor domain is known to function as an initiator for oligomerization of the Rep proteins. The cooperative effect of both domains appears to be further regulated by ATP binding as well as different DNA substrates such as dsDNA and ssDNA. Assembly of different nucleoprotein structures suggest that viral replication and genome integration is regulated and controlled by distinct Rep complexes which means that in presence of dsDNA Rep68 assembles to smaller complexes than in presence of ssDNA resulting in octamers.
Rep52 Involved in genome encapsidation Rep 52 is under the control of the p19 promoter and shares the same N-terminus with Rep78. It was shown that Rep52 possesses helicase and ATPase activity with 3´-5´polarity (Smith & Kotin, 1998). Despite the helicase activity, Rep52 and Rep78 share a putative zinc-finger domain, which suggest interactions with diverse cellular factors (Nash, Chen, Salganik, & Muzyczka, 2009) such as transcription factors (Lackner & Muzyczka, 2002) and TATA-binding proteins (Hermonat, Santin, Batchu, & Zhan, 1998).
Rep40
The smallest Rep protein (Rep40) possesses helicase and ATPase activity as well, but does not have strict requirements for DNA duplexes containing a 3´single-stranded end. Rep40 helicase activity requires bivalent ions such as Mg2+ or Mn2+ and is most active using ATP as substrate. Lacking the zinc finger domain, present in Rep52, Rep40 requires dimerization for functional helicase activity (Collaco, Kalman-Maltese, Smith, Dignam, & Trempe, 2003). Rep40/52 proteins are required for translocation of the single-stranded, viral genomes into the preformed capsids proceeding with the 3´end of the DNA (King, Dubielzig, Grimm, & Kleinschmidt, 2001).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 3611
  Illegal XhoI site found at 1913
  Illegal XhoI site found at 2099
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 4137
  Illegal SapI site found at 3048