Part:BBa_K404090

[AAV2]-Rep40ex

Rep 40
BioBrick Nr.	BBa_K404090
RFC standard	RFC 10
Requirement	pSB1C3
Source	pAAV_MCS
Submitted by	[http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]

Rep proteins

Overview

The Adeno-associated virus (AAV) consists of two open reading frames (ORF), rep and cap ORF. The four non-structural rep genes are driven by two promoters located at map units 5 (p5 promoter) and 19 (p19 promoter). Rep proteins are involved in genome encapsidation, regulation of gene expression and replication of the viral genome.

Figure 8: Genomic organization of the AAV-2 genome. The rep gene codes for four non-structural proteins – Rep40, Rep52, Rep68 and Rep78 – which are involved in gene regulation, genime encapsiation and viral DNA integration.

 

The two larger proteins Rep78/68 play an essential role in viral genome integration and regulation of AAV gene expression, whereas the smaller Rep proteins are involved in viral genome encapsidation. Rep proteins act both as repressors and activators of AAV transcription in respect to the absence and presence of helper viruses such as adenoviruses (Ad) or herpes simplex viruses (HSV) by interacting with several cellular proteins (Nash, Chen, Salganik, & Muzyczka, 2009).

Furthermore, in the absence of Rep proteins, as it is the case in recombinant AAVs, integration of the viral genome into the human genome is rare and random. There are several hotspots for integration of wtAAV genomes such as the human chromosome 19q13.42, known as the AAVSI site, but as well some other accessible chromatin regions for preferred integration have been found (5p13.3 and 3p24.3). Integration into the human genome is mediated by the two regulatory proteins Rep68 and Rep78 driven by the AAV p5 promoter. The proteins bind to the Rep binding site (RBS) which is located within the inverted terminal repeats (ITRs). The minimal consensus Rep binding site (RBS) GAGT GAGC is found within the ITRs and in the p5 integration-efficient element (p5IEE) of the p5 promoter (Hüser et al., 2010). Rep78/68 proteins possess DNA-binding, helicase and site-specific endonuclease activity located within the first 200 amino acids (Davis, Wu, & Owens, 2000). Since the N-terminal region is unique to the larger Rep proteins, the two smaller Rep proteins possess other biological functions. Rep52/40 gene expression is driven by the p19 promoter which is located within rep ORF and the proteins are involved in encapsidating the viral genome into the preformed capsids. Gene expression of these proteins is suppressed in absence of adenovirus infection by binding of Rep78/68 to the p5 promoter. Gene expression of p19 and p40 is transacvtivated by the Rep proteins Rep78/68 during coinfection.

Rep40

Rep40 in some minutes

40 kDa

ATPase and helicase activity

Involved in genome encapsidation

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).

Figure 9: Crystal structure of the SF-3 helicase (PDB: 1SH9).

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 53
  Illegal XhoI site found at 920
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]